3-Nitrogen-6,7-dioxygen steroids and uses related thereto

ABSTRACT

A compound of formula (I) and pharmaceutical acceptable salts, solvates, stereoisomers and prodrugs thereof, in isolation or in mixture, wherein, independently at each occurrence: R 1  and R 2  are selected from hydrogen, oxygen so as to form nitro or oxime, amino, sulfate, and sulfonic acid, and organic groups having 1-30 carbons and optionally containing 1-6 heteroatoms selected from nitrogen, oxygen, phosphorous, silicon, and sulfur, where R 1  and R 2  may, together with the N to which they are both bonded, form a heterocyclic structure that may be part of an organic group having 1-30 carbons and optionally containing 1-6 heteroatoms selected from nitrogen, oxygen and silicon, and where R 1  may be a 2, or 3 atom chain to numeral 2 so that —N—R 1 — forms part of a fused bicyclic structure to ring A; R 3  and R 4  are selected from direct bonds to 6 and 7 respectively so as to form carbonyl groups, hydrogen, or a protecting group such that R 3  and/or R 4  is part of hydroxyl or carbonyl protecting group; numerals 1 through 17 each represent a carbon having substitution as described. The compounds may be formulated into pharmaceutical compositions, and used in the treatment and/or prevention of various conditions, including inflammation, asthma, an allergic disease, chronic obstructive pulmonary disease, atopic dermatitis, solid tumors, AIDS, ischemia, and cardiac arrhythmias.

TECHNICAL FIELD

[0001] This invention is directed towards 3-amino-6,7-dioxygenatedsteroids, compositions including these steroids, and therapeutic usesrelated thereto.

BACKGROUND OF THE INVENTION The Inflammatory Response (Inflammation)

[0002] Inflammation is an essential localized host response to invadingmicroorganisms or tissue injury which involves cells of the immunesystem. The classic signs of inflammation include redness (erythema),swelling (edema), pain and increased heat production (pyrema) at thesite of injury. The inflammatory response allows the body tospecifically recognize and eliminate an invading organism and/or repairtissue injury. Many of the acute changes at the site of inflammation areeither directly or indirectly attributable to the massive influx ofleukocytes (e.g. neutrophils, eosinophils, lymphocytes, monocytes) whichis intrinsic to this response. Leukocytic infiltration and accumulationin tissue results in their activation and subsequent release ofinflammatory mediators such as LTB₄, prostaglandins, TNF-α, IL-1β, IL-8,IL5, IL4, histamine, proteases and reactive oxygen species for example.

[0003] Normal inflammation is a highly regulated process that is tightlycontrolled at several levels for each of the cell types involved in theresponse. For example, expression of the pro-inflammatory cytokine TNF-αis controlled at the level of gene expression, translation,post-translational modification and release of the mature form from thecell membrane. Many of the proteins up-regulated during inflammation arecontrolled by the transcription factor, NF-κB. Pro-inflammatoryresponses are countered in some instances by endogenousanti-inflammatory mechanisms such as generation of IL-10. Acharacteristic of a normal inflammatory response is that it is temporaryin nature and is followed by a resolution phase which brings the stateof the tissue back to its prior condition. The resolution phase isthought to involve up-regulation of anti-inflammatory mechanisms, suchas IL-10, as well as down-regulation of the pro-inflammatory processes.

Inflammatory Disease

[0004] Inflammatory disease occurs when an inflammatory response isinitiated that is inappropriate and/or does not resolve in the normalmanner but rather persists and results in a chronic inflammatory state.Inflammatory disease may be systemic (e.g., lupus) or localized toparticular tissues or organs and exerts an enormous personal andeconomic burden on society. Examples of some of the most common andproblematic inflammatory diseases include asthma, allergy, rheumatoidarthritis, inflammatory bowel disease, psoriasis, emphysema, colitis,graft vs host disease, contact dermatitis, and ischemia-reperfusioninjury. Other disease states such as immunodeficiency diseases are nowknown to be associated with altered regulation of the chemokine/cytokinenetwork and their receptors, which can alter viral replication and AIDSpathogenesis.

[0005] Many of the tissue, cellular and biochemical processes which areperturbed in inflammatory disease have been elucidated and this hasallowed the development of experimental models or assays to mimic thedisease state. These in-vitro and in-vivo assays enable selection andscreening of compounds with a high probability of therapeutic efficacyin the relevant inflammatory disease. For example, the ability of acompound to inhibit the allergen-induced accumulation of inflammatorycells such as eosinophils and lymphocytes in the lavage fluid obtainedfrom sensitized animals is indicative of anti-asthma activity. Inparticular, this model system is useful in the evaluation of the effectsof compounds in the treatment of the late phase response andhyper-responsiveness that is characteristic of asthma, when lunginflammation is apparent.

Asthma and Allergy

[0006] Asthma and allergy are closely related with good evidence fromclinical studies demonstrating a strong correlation between the severityof asthma and the degree of atopy (allergy). Sensitization to allergensis believed to be the most important risk factor for asthma in bothchildren and adults, with approximately 90% of asthma cases exhibitingatopy.

[0007] Allergy is characterized by an increased blood serum IgE(antibody) level. Repeated exposure to allergens, in a process calledsensitization, is normally required to trigger atopy and the subsequentasthmatic or allergic response. Once B cells are exposed to allergens,they produce antibodies which bind to the surface of mast cells. Thecrosslinking of two antibodies by the antigen causes a series ofreactions resulting in degranulation and the release of a number ofmediators which modulate the inflammatory response. Mediators that arereleased or generated during the asthmatic and allergic response includehistamine, leukotrienes, prostaglandins, cytoldnes and tryptase.

[0008] Asthma is characterized by hyperresponsiveness of the airways,episodic periods of bronchospasm and chronic inflammation of the lungs.Obstruction of the airways is reversible with time or in response todrug therapies. Patients exhibiting normal airflow may be hyperreactiveto a variety of naturally occurring stimuli, e.g., cold air, exercise,chemicals and allergen. The most common event initiating an asthmaticresponse is an immediate hypersensitivity to common allergens includingragweed pollen, grass pollen, various fungi, dust mites, cockroaches anddomestic animals. The symptoms of the disease include chest tightness,wheezing, shortness of breath and coughing. Asthma incidence andmortality has been increasing worldwide, doubling over the past 20 yearsdespite modem therapies.

[0009] The responses of the airways to allergen is complex and consistsof an early asthmatic response (EAR) which peaks 20-30 min afterexposure to the stimuli, is characterized by bronchoconstriction andnormally resolves after 1½ to 2 hours. The late asthmatic response (LAR)generally occurs 3-8 hours after initial exposure, and involves bothbronchoconstriction and the development of inflammation and edema in thelung tissue. This inflammation often becomes chronic, with epithelialdamage occurring and infiltration of the lungs with inflammatory cellssuch as eosinophils and neutrophils.

Current Treatments for Asthma

[0010] Glucocorticoids (steroids) are the most effective long-termtherapy for the treatment of asthma. For example, due to the presence ofairway inflammation even in mild asthma, inhaled steroids are used evenin early stage drug therapy. Although steroids are effectiveanti-inflammatories they are not very useful for the control of acuteasthma attacks. Orally delivered steroids are associated withsignificant side-effects and consequently their chronic use in thecontrol of asthma is minimal. Combination therapy is often employed fororally delivered steroids, where combination therapy may be divided intothe following areas: anti-inflammatory drugs (e.g., inhaled and oralsteroids), bronchodilators, (e.g., β₂-agonists, xantlines,anticholinergics), and mediator inhibitors (e.g., cromolyns andleukotriene antagonists). In general, moderate to severe asthma patientsare poorly served by the present armamentarium of drugs. Drugs that aresafe are only marginally effective, while effective drugs haveunacceptable side effects with extensive monitoring of patientsrequired. Products under development continue to meet challenges relatedto side-effects (e.g., emesis side-effects characteristic of certainphosphodiesterase 4 inhibitors) and poor pharmacokinetic and metabolismparameters. There is a significant need for therapeutic agents thatachieve safe and effective treatment of inflammatory diseases such asasthma and allergy. The present invention provides these and relatedbenefits as described herein.

SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides compounds accordingto formula (1) and pharmaceutically acceptable salts, solvates,stereoisomers and prodrugs thereof, in isolation or in mixtures,

[0012] wherein, independently at each occurrence:

[0013] R¹ and R² are selected from hydrogen, oxygen so as to form nitroor oxime, amino, —SO₃—R, and organic groups having 1-30 carbons andoptionally containing 1-6 heteroatoms selected from nitrogen, oxygen,phosphorous, silicon, and sulfur, where R² may be a direct bond tonumeral 3, or R¹ and R² may, together with the N to which they are bothbonded, form a heterocyclic structure that may be part of an organicgroup having 1-30 carbons and optionally containing 1-6 heteroatomsselected from nitrogen, oxygen and silicon; and where R¹ may be a 2, or3 atom chain to numeral 2 so that —N—R¹— forms part of a fused bicyclicstructure to ring A;

[0014] R³ and R⁴ are selected from direct bonds to 6 and 7 respectivelyso as to form carbonyl groups, hydrogen, or a protecting group such thatR³ and/or R⁴ is part of hydroxyl or carbonyl protecting group;

[0015] numerals 1 through 17 each represent a carbon, where carbons atnumerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independently substitutedwith

[0016] (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or

[0017] (b) two of the following, which are independently selected: —X,—N(R¹)(R²), —R⁵ and —OR⁶;

[0018] and where carbons at numerals 5, 8, 9, 10, 13 and 14 may beindependently substituted with one of —X, —R⁵, —N(R¹)(R²) or —OR⁶;

[0019] in addition to the —OR³ and —OR⁴ groups as shown, each of carbons6 and 7 may be independently substituted with one of —X, —N(R¹)(R²), —Ror —OR⁶;

[0020] each of rings A, B, C and D is independently fully saturated,partially saturated or fully unsaturated;

[0021] R⁵ at each occurrence is independently selected from H, X, andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded;

[0022] R⁶ is H or a protecting group such that —OR⁶ is a protectedhydroxyl group, where vicinal —OR⁶ groups may together form a cyclicstructure that protects vicinal hydroxyl groups, and where geminal —OR⁶groups may together form a cyclic structure that protects a carbonylgroup; and

[0023] X represents fluoride, chloride, bromide and iodide.

[0024] In another aspect, the present invention provides apharmaceutical composition comprising a steroid compound as set forthabove, and a pharmaceutically acceptable carrier, excipient or diluent.

[0025] In another aspect, the present invention provides a method oftreating inflammation comprising administering to a subject in needthereof a therapeutically-effective amount of a steroid compound as setforth above.

[0026] In another aspect, the present invention provides a method oftreating inflammation prophylactically comprising administering to asubject in need thereof a prophylactically-effective amount of a steroidcompound as set forth above.

[0027] In another aspect, the present invention provides a method oftreating asthma comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

[0028] In another aspect, the present invention provides a method oftreating allergic disease including but not limited to dermal and ocularindications comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

[0029] In another aspect, the present invention provides a method oftreating chronic obstructive pulmonary disease comprising administeringto a subject in need thereof a therapeutically-effective amount of asteroid compound as set forth above.

[0030] In another aspect, the present invention provides a method oftreating atopic dermatitis comprising administering to a subject in needthereof a therapeutically-effective amount of a steroid compound as setforth above.

[0031] In another aspect, the present invention provides a method oftreating solid tumours comprising administering to a subject in needthereof a therapeutically-effective amount of a steroid compound as setforth above.

[0032] In another aspect, the present invention provides a method oftreating AIDS comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

[0033] In another aspect, the present invention provides a method oftreating ischemia reperfusion injury comprising administering to asubject in need thereof a therapeutically-effective amount of a steroidcompound as set forth above.

[0034] In another aspect, the present invention provides a method oftreating cardiac arrhythmias comprising administering to a subject inneed thereof a therapeutically-effective amount of a steroid compound asset forth above.

[0035] These and related aspects of the present invention are describedin more detail below.

BRIEF DESCRIPTION OF THE DRAWING

[0036]FIGS. 1A and 1B depict summaries of synthetic transformations thatmay be used to convert a 3-amino steroid into a 3-nitrogen steroid ofthe present invention.

[0037]FIGS. 2A, 2B and 2C are a set of bar graphs showing the effect ofcompound 89 (dose response, 4 doses qd, p.o.) on ovalbumin-inducedaccumulation of inflammatory cells in the lung lavage fluid obtainedfrom sensitized Brown Norway rats. FIG. 2A shows the accumulation ofeosinophils, FIG. 2B shows the accumulation of neutrophils, and FIG. 2Cshows the accumulation of lymphocytes.

[0038]FIGS. 3A, 3B and 3C are a set of bar graphs showing the effect ofcompound 28 (dose response, 4 doses qd, p.o.) on ovalbumin-inducedaccumulation of inflammatory cells in the lung lavage fluid obtainedfrom sensitized Brown Norway rats. FIG. 3A shows the accumulation ofeosinophils, FIG. 3B shows the accumulation of neutrophils, and FIG. 3Cshows the accumulation of lymphocytes.

[0039]FIG. 4 is a graph showing the effect of test compounds 28 and 89,administered orally once per day for 4 days prior to challenge, onallergen-induced changes in lung resistance in sensitized guinea pigs.

[0040]FIG. 5 is a graph showing the effect of test compounds 28 and 89,administered orally once per day for 4 days prior to challenge, onallergen-induced changes in lung elastance in sensitized guinea pigs.

[0041]FIG. 6 is a graph showing duration of anti-bronchospastic activityof test compound 89, administered orally at 1 mg/kg once per day for 4days prior to challenge, on allergen-induced changes in lung resistancein sensitized guinea pigs.

[0042]FIG. 7 is a graph showing duration of anti-bronchospastic activityof test compound 89, administered orally at 1 mg/kg once per day for 4days prior to challenge, on allergen-induced changes in lung elastancein sensitized guinea pigs.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention provides compounds, compositions andmethods useful in the treatment and/or prevention of various diseaseconditions. For example, in one aspect, the present invention provides amethod of treating and/or preventing an inflammatory disease. The methodincludes administering to a subject in need thereof an effective amountof a compound of formula (1) or pharmaceutically acceptable salt,solvate, stereoisomer or prodrug thereof, or an effective amount of acomposition containing a compound of formula (1) or pharmaceuticallyacceptable salt, solvate, stereoisomer or prodrug thereof.

[0044] Before describing the invention in further detail, certaindefinitions as used herein are provided with the following definitions,and certain conventions used herein are also set forth.

[0045] A. Definition of Terms

[0046] As used herein, the following terms have the indicated meaning,unless clearly indicated otherwise.

[0047] “Alkyl” is a monovalent, saturated or unsaturated, straight,branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. Invarious embodiments, the alkyl group has 1-20 carbon atoms, i.e., is aC1-C20 (or C₁-C₂₀) group, or is a C1-C18 group, a C1-C12 group, a C1-C6group, or a C1-C4 group. Independently, in various embodiments, thealkyl group: has zero branches (i.e., is a straight chain), one branch,two branches, or more than two branches; is saturated; is unsaturated(where an unsaturated alkyl group may have one double bond, two doublebonds, more than two double bonds, and/or one triple bond, two triplebonds, or more than two triple bonds); is, or includes, a cyclicstructure; is acyclic. Exemplary alkyl groups include C₁alkyl (i.e.,—CH₃ (methyl)), C₂alkyl (i.e., —CH₂CH₃ (ethyl), —CH═CH₂ (ethenyl) and—C≡CH (ethynyl)) and C₃alkyl (i.e., —CH₂CH₂CH₃ (n-propyl), —CH(CH₃)₂(i-propyl), —CH═CH—CH₃ (1-propenyl), —C≡C—CH₃ (1-propynyl), —CH₂—CH═CH₂(2-propenyl), —CH₂—C≡CH (2-propynyl), —C(CH₃)═CH₂ (1-methylethenyl), and—CH(CH₂)₂ (cyclopropyl)).

[0048] “Aryl” is a monovalent, aromatic, hydrocarbon, ring system. Thering system may be monocyclic or fused polycyclic (e.g., bicyclic,tricyclic, etc.). In various embodiments, the monocyclic aryl ring isC5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to thenumber of carbon atoms that form the ring system. A C6 ring system,i.e., a phenyl ring, is a preferred aryl group. In various embodiments,the polycyclic ring is a bicyclic aryl group, where preferred bicyclicaryl groups are C8-C12, or C9-C10. A naphthyl ring, which has 10 carbonatoms, is a preferred polycyclic aryl group.

[0049] “Heteroalkyl” is an alkyl group (as defined herein) wherein atleast one of the carbon atoms is replaced with a heteroatom. Preferredheteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may,but typically does not, have the same number of valence sites as carbon.Accordingly, when a carbon is replaced with a heteroatom, the number ofhydrogens bonded to the heteroatom may need to be increased or decreasedto match the number of valence sites of the heteroatom. For instance, ifcarbon (valence of four) is replaced with nitrogen (valence of three),then one of the hydrogens formerly attached to the replaced carbon mustbe deleted. Likewise, if carbon is replaced with halogen (valence ofone), then three (i.e., all) of the hydrogens formerly bonded to thereplaced carbon must be deleted.

[0050] “Heteroaryl” is a monovalent aromatic ring system containingcarbon and at least one heteroatom in the ring. The heteroaryl groupmay, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may bemonocyclic or polycyclic, where the polycyclic ring may contained fused,spiro or bridged ring junctions. In one embodiment, the heteroaryl isselected from monocyclic and bicyclic. Monocyclic heteroaryl rings maycontain from about 5 to about 10 member atoms (carbon and heteroatoms),preferably from 5-7, and most preferably from 5-6 member atoms in thering. Bicyclic heteroaryl rings may contain from about 8-12 memberatoms, or 9-10 member atoms in the ring. The heteroaryl ring may beunsubstituted or substituted. In one embodiment, the heteroaryl ring isunsubstituted. In another embodiment, the heteroaryl ring issubstituted. Exemplary heteroaryl groups include benzofuran,benzothiophene, furan, imidazole, indole, isothiazole, oxazole,piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, quinoline, thiazole and thiophene.

[0051] “Heteroatom” is a halogen, nitrogen, oxygen, phosphorous, siliconor sulfur atom. Groups containing more than one heteroatom may containdifferent heteroatoms.

[0052] “Hydrocarbons” are chemical groups formed exclusively of hydrogenand carbon; “Halocarbons” are chemical groups formed exclusively ofhalogen and carbon; and “Hydrohalocarbons” are chemical groups formedexclusively of hydrogen, halogen, and carbon.

[0053] “Organic groups” and “Organic moieties” are used synonymously,and refer to stable structures having the indicated number and type ofatoms.

[0054] “Pharmaceutically acceptable salt” and “salts thereof” in thecompounds of the present invention refers to acid addition salts andbase addition salts.

[0055] Acid addition salts refer to those salts formed from compounds ofthe present invention and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

[0056] Base addition salts refer to those salts formed from compounds ofthe present invention and inorganic bases such as sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum salts and the like. Suitable salts include the ammonium,potassium, sodium, calcium and magnesium salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

[0057] When any variable occurs more than one time in any constituent orin compounds of formula (1), its definition on each occurrence isindependent of its definition at every other occurrence. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds. The compounds useful in the methods andcompositions of the present invention, as well as the compounds of thepresent invention, may have asymmetric centers and occur as racemates,racemic mixtures and as individual diastereomers, or enantiomers withall isomeric forms being included in the present invention. A racemateor racemic mixture does not imply a 50:50 mixture of stereoisomers.

[0058] In another embodiment, the present invention providespharmaceutical compositions containing a compound of formula (1) as setforth above, in combination with a pharmaceutically-acceptable carrier,diluent or excipient. These compositions may be used for the treatmentof inflammation or other conditions as disclosed herein. Thesecompositions may also be formed into a medicament, which may be used inthe treatment of, for example, inflammation.

[0059] These compositions are useful as, for example, assay standards,convenient means of making bulk shipments, or pharmaceuticalcompositions. An assayable amount of a compound of the invention is anamount which is readily measurable by standard assay procedures andtechniques as are well known and appreciated by those skilled in theart. Assayable amounts of a compound of the invention will generallyvary from about 0.001 wt % to about 100 wt % of the entire weight of thecomposition. Inert carriers include any material which does not degradeor otherwise covalently react with a compound of formula (1). Examplesof suitable inert carriers are water; aqueous buffers, such as thosewhich are generally useful in High Performance Liquid Chromatography(HPLC) analysis; organic solvents, such as acetonitrile, ethyl acetate,hexane and the like; and pharmaceutically acceptable carriers.

[0060] “Pharmaceutically acceptable carriers” for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

[0061] Steroid compounds of the invention have at least four rings,commonly designated as A, B, C and D as shown below, where ring A may befused to an additional ring:

[0062] B. Compounds

[0063] The present invention provides compounds according to formula (1)and pharmaceutically acceptable salts, solvates, stereoisomers andprodrugs thereof, in isolation or in mixtures,

[0064] wherein, independently at each occurrence:

[0065] R¹ and R² are selected from hydrogen, oxygen so as to form nitroor oxime, amino, —SO₃—R, and organic groups having 1-30 carbons andoptionally containing 1-6 heteroatoms selected from nitrogen, oxygen,phosphorous, silicon, and sulfur, where R² may be a direct bond tonumeral 3, or R¹ and R² may, together with the N to which they are bothbonded, form a heterocyclic structure that may be part of an organicgroup having 1-30 carbons and optionally containing 1-6 heteroatomsselected from nitrogen, oxygen and silicon, and where R¹ may be a 2, or3 atom chain to numeral 2 so that —N—R¹— forms part of a fused bicyclicstructure to ring A;

[0066] R³ and R⁴ are selected from direct bonds to 6 and 7 respectivelyso as to form carbonyl groups, hydrogen, or a protecting group such thatR³ and/or R⁴ is part of hydroxyl or carbonyl protecting group;

[0067] numerals 1 through 17 each represent a carbon, where carbons atnumerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independently substitutedwith

[0068] (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or

[0069] (b) two of the following, which are independently selected: —X,—N(R¹)(R²), —R⁵ and —OR⁶;

[0070] and where carbons at numerals 5, 8, 9, 10, 13 and 14 may beindependently substituted with one of —X, —R⁵, —N(R¹)(R²) or —OR⁶;

[0071] in addition to the —OR³ and —OR⁴ groups as shown, each of carbons6 and 7 may be independently substituted with one of —X, —N(R¹)(R²), —R⁵or —OR⁶;

[0072] each of rings A, B, C and D is independently fully saturated,partially saturated or fully unsaturated;

[0073] R⁵ at each occurrence is independently selected from H, X, andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded;

[0074] R⁶ is H or a protecting group such that —OR⁶ is a protectedhydroxyl group, where vicinal —OR⁶ groups may together form a cyclicstructure that protects vicinal hydroxyl groups, and where geminal —OR⁶groups may together form a cyclic structure that protects a carbonylgroup; and

[0075] X represents fluoride, chloride, bromide and iodide.

[0076] In one aspect of the invention, R¹ and R² are selected fromhydrogen and organic groups having 1-30 carbons and optionallycontaining 1-6 heteroatoms selected from nitrogen, oxygen, phosphorous,silicon, and sulfur. Optionally, R² is a direct bond to numeral 3. Inanother aspect, R¹, R², and the N to which they are both bonded, form aheterocyclic structure that may be part of an organic group having 1-30carbons and optionally containing 1-6 heteroatoms selected fromnitrogen, oxygen and silicon. In another aspect, R¹ is a 2, or 3 atomchain to numeral 2 so that —N—R¹— forms part of a fused bicyclicstructure to ring A, and the 2 or 3 atoms are selected from C, N and O,so long as a stable structure results. Optionally, in these and otheraspects of the present invention, the organic group has 1-20 carbons,while in another optional embodiment the organic group has 1-10 carbons.

[0077] In a preferred aspect of the invention, each of R¹ and R² ishydrogen. These steroids not only have desirable biological activity,they also serve as convenient precursor compounds to preparing othersteroid of the invention wherein R¹ and/or R² is not hydrogen.

[0078] For example, in one embodiment, the invention provides a compoundof formula (1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selectedfrom direct bonds to 6 and 7 respectively so as to form carbonyl groups,hydrogen, or a protecting group such that R³ and/or R⁴ is part ofhydroxyl or carbonyl protecting group; and in addition to the —OR³ and—OR⁴ groups as shown, each of carbons 6 and 7 is substituted withhydrogen unless precluded because —OR³ or —OR⁴ represent a carbonylgroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═O, ═C(R⁵)(R⁵), ═C═(R⁵)(R⁵), —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and—(O(C(R⁵)(R⁵))_(n)O)— wherein n ranges from 1 to about 6; or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), —R⁵ and—OR⁶; each of rings A, B, C and D is independently fully saturated,partially saturated or fully unsaturated; R⁵ at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, oxygen, silicon and sulfur;where two geminal R⁵ groups may together form a ring with the carbonatom to which they are both bonded; R⁶ is H or a protecting group suchthat —OR⁶ is a protected hydroxyl group, where vicinal —OR⁶ groups maytogether form a cyclic structure that protects vicinal hydroxyl groups,and where geminal —OR⁶ groups may together form a cyclic structure thatprotects a carbonyl group; and X represents fluoride, chloride, bromideand iodide.

[0079] In another one embodiment, the invention provides a compound offormula (1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selected fromhydrogen and protecting groups such that R³ and/or R⁴ is part ofhydroxyl protecting group; carbons at numerals 1, 2, 4, 11, 12, 15 and16 are each substituted with two hydrogens; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), and—R⁵; each of rings A, B, C and D is independently fully saturated orpartially saturated; R⁵ at each occurrence is independently selectedfrom H, X, and C₁₋₃₀ hydrocarbons, halocarbons and halohydrocarbons; andX represents fluoride, chloride, bromide and iodide.

[0080] In another one embodiment, the invention provides a compound offormula (1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selected fromhydrogen and protecting groups such that R³ and/or R⁴ is part ofhydroxyl protecting group; carbons at numerals 1, 2, 4, 11, 12, 15 and16 are each substituted with two hydrogens; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of—R⁵; each of rings A, B, C and D is independently fully saturated orpartially saturated; and R⁵ at each occurrence is independently selectedfrom H and C₁₋₁₀ hydrocarbons.

[0081] Specific compounds of the present invention wherein R¹ and R² arehydrogen include:

[0082] In another aspect, the invention provides steroids having3-nitrogen substitution, where the 3-nitrogen is substituted with anorganic group. For instance, the invention provides steroid compoundswherein R¹ is selected from—C(═O)—R⁷, —C(═O)NH—R⁷; and —SO₂—R⁷; whereinR⁷ is selected from alkyl, heteroalkyl, aryl and heteroaryl groups. In arelated embodiment, R¹ is hydrogen and R² is —CH₂—R⁷ wherein R⁷ isselected from alkyl, heteroalkyl, aryl and heteroaryl. In oneembodiment, R⁷ is selected from C₁₋₁₀hydrocarbyl. In another embodiment,—C(═O)—R⁷ comprises biotin. In another embodiment, R⁷ is selected fromalkyl-substituted phenyl; halogen-substituted phenyl; alkoxy-substitutedphenyl; aryloxy-substituted phenyl; and nitro-substituted phenyl.

[0083] In another aspect, (R¹)²)N— is a heterocycle, that is, the N of(R¹)(R²)N— may be part of a heterocyclic ring. Examples include:

[0084] In another aspect, either or both of R¹ and R² comprises aheterocyclic ring or a carbocyclic ring. A preferred heterocyclic ringis

[0085] and a preferred carbocyclic ring is phenyl, which includessubstituted phenyl such as 3-methylphenyl; 4-hydroxyphenyl; and4-sulfonamidephenyl.

[0086] In another aspect, R¹ may be a 2, or 3 atom chain to numeral 2 sothat —N—R¹— forms part of a fused bicyclic structure to ring A. Thus,the present invention provides compounds of the formula shown below,where Z represents 2 or 3 atoms, selected from C, N and O. The ringincluding Z may be saturated or unsaturated.

[0087] Examples of such fused ring compounds include:

[0088] In another aspect, R¹ is hydrogen and R² comprises aC₁₋₁₀hydrocarbyl.

[0089] In another aspect, R¹ is hydrogen and R² is heteroalkyl. Suitableheteroalkyl include, without limitation,C₁₋₁₀alkyl-W—C₁₋₁₀alkylene-wherein W is selected from O and NH;HO—C₁₋₁₀alkylene-; and HO—C₁₋₁₀alkylene-W—C₁₋₁₀alkylene-where W isselected from O and NH.

[0090] In another aspect, each of R¹ and R² is independently selectedfrom hydrogen and organic groups having 1-20 carbons and optionallycontaining 1-5 heteroatoms selected from nitrogen, oxygen, silicon, andsulfur.

[0091] In another aspect, each of R¹ and R² is independently selectedfrom hydrogen, R⁸, R⁹, R¹⁰, R¹¹ and R¹² where R⁸ is selected fromC₁₋₁₀alkyl, C₁₋₁₀heteroalkyl comprising 1, 2 or 3 heteroatoms, C₆₋₁₀aryland C₃₋₁₅heteroaryl comprising 1, 2 or 3 heteroatoms; R⁹ is selectedfrom (R⁸)_(r)—C₁₋₁₀alkylene, (R⁸)_(r)—C₁₋₁₀heteroalkylene comprising 1,2 or 3 heteroatoms, (R⁸)_(r)—C₆₋₁₀arylene and(R⁸)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹⁰ isselected from (R⁹)_(r)—C₁₋₁₀alkylene, (R⁹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁹)_(r)—C₆₋₁₀arylene, and(R⁹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹¹ isselected from (R¹⁰)_(r)—C₁₋₁₀aloylene, (R¹⁰)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹⁰)_(r)—C₆₋₁₀arylene, and(R¹⁰)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, R¹² isselected from (R¹¹)_(r)—C₁₋₁₀alkylene, (R¹¹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹¹)_(r)—C₆₋₁₀arylene, and(R¹¹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, and r isselected from 0, 1, 2, 3, 4 and 5, with the proviso that R¹ and R² mayjoin to a common atom so as to form a ring with the common atom.

[0092] In another aspect, the present invention provides steroidcompounds of the structure shown above, wherein: R¹ and R² areindependently selected from hydrogen, R⁸, R⁹, R¹⁰, R¹¹ and R¹² where R⁸is selected from alkyl, heteroalkyl, aryl and heteroaryl; R⁹ is selectedfrom (R⁸)_(r)-alkylene, (R⁸)_(r)-heteroalkylene, (R⁸)_(r)-arylene and(R⁸)_(r)-heteroarylene; R¹⁰ is selected from (R⁹)_(r)-alkylene,(R⁹)_(r)-heteroalkylene, (R⁹)_(r)-arylene, and (R⁹)_(r)-heteroarylene;R¹¹ is selected from (R¹⁰)_(r)-alkylene, (R¹⁰)_(r)-heteroalkylene,(R¹⁰)_(r)-arylene, and (R¹⁰)_(r)-heteroarylene, R¹² is selected from(R¹¹)_(r)-alkylene, (R¹¹)_(r)-heteroalkylene, (R¹¹)_(r)-arylene, and(R¹¹)_(r)-heteroarylene, and r is selected from 0, 1, 2, 3, 4 and 5,with the proviso that R¹ and R² may join to a common atom so as to forma ring with the common atom; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of —R⁵; each ofrings A, B, C and D is independently fully saturated or partiallysaturated; and R⁵ at each occurrence is independently selected from Hand C₁₋₁₀ hydrocarbons.

[0093] For instance, R¹ and R² are selected from hydrogen, CH₃—,CH₃(CH₂)₂—, CH₃(CH₂)₄—, CH₃CO—, C₆H₅CO—(CH₃)₂CHSO₂—, C₆H₅SO₂—,C₆H₅NHCO—, CH₃(CH₂)₂NHCO—, CH₃(CH₂)₂NH(CH₂)₂—, (CH₃)₂N(CH₂)₂—,HOCH₂CH₂—, HOCH₂(CH₂)₄—, HOCH₂CH₂NHCH₂CH₂—, 3-(CH₃)C₆H₄—, 4-(HO)C₆H₄—,4-(H₂NSO₂)C₆H₄—, 4-((CH₃)₂CH)C₆H₄—CH₂—, 2-(C₆H₄—CH₂—, 3-(CF₃)C₆H₄—CH₂—,2-(CH₃O)C₆H₄—CH₂—, 4-(CF₃O)C₆H₄—CH₂—, 3-(C₆H₅O)C₆H₄—CH₂—,3-(NO₂)C₆H₄—CH₂—,

[0094] to which they are both attached and form a heterocycle selectedfrom:

[0095] Specific compounds of the present invention wherein R¹ ishydrogen but R² is not hydrogen include:

[0096] Thus, one set of preferred compounds of the invention have R¹equal to hydrogen but R² is not equal to hydrogen.

[0097] In steroid compounds of the invention as disclosed above, in oneaspect each of R³ and R⁴ is hydrogen, i.e., the steroid has hydroxysubstitution at each of the carbons located at numerals 6 and 7. In arelated aspect, one or both of the hydroxy groups at carbons 6 and 7 arein a protected form, ie., are bonded to a hydroxy protecting group. Suchprotecting groups are well known in the art, and are disclosed in, e.g.,Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley &,Sons, New York, N.Y. (1999). A suitable protecting group is a ketal, sothat the present invention provides compounds of the structure:

[0098] As stated above, the present invention provides steroid compoundsthat include compounds of defined stereochemistry. One such compound hasthe stereochemistry shown in the following structure for R³O— and R⁴O—:

[0099] As also stated above, the present invention provides salt formsof the steroids of the present invention, preferably pharmaceuticallyacceptable salts. In one embodiment, —N(R¹)(R²) is in a salt form. Inother words, —N(R¹)(R²) is protonated so that the N carries a positivecharge. In such a case, the steroid compound of the present invention isan acid addition salt as defined herein. In a preferred aspect, thepresent invention provides hydrochloride salts of the steroid structuresshown above. In another preferred aspect, the present invention providesacetate salts of the steroid structures shown herein.

[0100] As also stated above, the present invention provides prodrugs ofthe specific compounds shown by formula (1). In one aspect, the presentinvention is directed to a prodrug of any of the specific compoundsshown by formula (1). In another aspect, the present invention excludesprodrugs of the specific compounds shown by formula (1), i.e., in oneaspect the present invention is directed to compounds of formula (1) andpharmaceutically acceptable salts, solvates, stereoisomers but notprodrugs thereof, in isolation or in mixture.

[0101] In steroid compounds of the invention as set forth above, in apreferred embodiment, 17 is substituted with ═C(R⁵)(R⁵) and R⁵ isselected from hydrogen, halogen, C₁₋₆alkyl, C₁₋₆ hydroxyallyl, and—CO₂—C₁₋₆alkyl. In other preferred embodiment, 17 is substituted withC₁₋₆alkyl or C₁₋₆haloalkyl; or 17 is substituted with —OR⁶ or ═O,wherein R⁶ is hydrogen.

[0102] In steroid compounds of the invention as set forth herein, in apreferred embodiment, at least one of 10 and 13 is substituted withmethyl.

[0103] In steroid compounds of the invention as set forth herein, in apreferred embodiment, numerals 1 through 16 each represent a carbon,where carbons at numerals 1, 2, 4, 11, 12, 15 and 16 may beindependently substituted with: (a) one of ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or (b) two of the following, which are independentlyselected: —X, —N(R¹)(R²), —R⁵ and —OR⁶; and numeral 17 represents acarbon substituted with: (a) one of: ═C(R^(5a))(^(5a)),═C═C(R^(5a))(R^(5a)), and═C═C(R^(5a))(R^(5a)), and—C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)— wherein n ranges from 1 toabout 6; or (b) two of the following, which are independently selected:—X, —N(R¹)(R²), and —R^(5a); where R^(5a) at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, silicon and sulfur; where twogeminal R⁵ groups may together form a ring with the carbon atom to whichthey are both bonded. Optionally, R^(5a) at each occurrence isindependently selected from C₁₋₃₀ hydrocarbon, C₁₋₃₀ halocarbon, C₁₋₃₀hydrohalocarbon, H, and X. In an alternative optional embodiment, R^(5a)at each occurrence is independently selected from C₁₋₁₀ hydrocarbon,C₁₋₁₀ halocarbon, C₁₋₁₀ hydrohalocarbon, H, and X. Optionally, in eachof these listed embodiment, the present invention provides a furtherembodiment wherein R¹ and R² are selected from hydrogen, oxygen so as toform nitro or oxime, amino, —SO₃—R, and organic groups having 1-30carbons and optionally containing 1-6 heteroatoms selected from oxygen,phosphorous, silicon, and sulfur, where R² may be a direct bond tonumeral 3, or R¹ and R² may, together with the N to which they are bothbonded, form a heterocyclic structure that may be part of an organicgroup having 1-30 carbons and optionally containing 1-6 heteroatomsselected from oxygen and silicon; or R¹ may be a 2 or 3 atom chain tonumeral 2 so that —N—R¹— forms part of a fused bicyclic structure toring A. Optionally, in each of these listed embodiments, the presentinvention provides a further embodiment wherein carbons at numerals 1,2, 4, 11, 12, 15 and 16 are each substituted with two hydrogens unlesssaid carbon is part of an unsaturated bond; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen unless said carbon is partof an unsaturated bond; carbon at numeral 10 is substituted with methyl;and carbon at number 13 is substituted with methyl unless it is part ofan unsaturated bond. Optionally, in each of these listed embodiments,the present invention provides a further embodiment wherein carbons atnumerals 1, 2, 4, 11, 12, 15 and 16 are each substituted with twohydrogens; carbons at numerals 5, 8, 9 and 14 are each substituted withone hydrogen; carbon at numeral 10 is substituted with methyl; andcarbon at number 13 is substituted with methyl unless it is part of anunsaturated bond.

[0104] In steroid compounds of the invention as set forth herein, in apreferred embodiment, each of R¹ and R² is hydrogen; and/or each of R³and R⁴ is hydrogen; and/or the carbon at numeral 17 is substituted with(a) one of the following: C(R^(5a)(R^(5a)), ═C═(R^(5a))(R^(5a)), and—C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)— wherein n ranges from 1 toabout 6; or (b) two of the following, which are independently selected:—X, —N(R¹)(R²) and —R^(5a); where R^(5a) at each occurrence isindependently selected from H, X and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, silicon and sulfur; where twogeminal R⁵ groups may together form a ring with the carbon atom to whichthey are both bonded.

[0105] In steroids of the present invention, unless otherwise indicated,each of rings A, B, C and D is independently fully saturated, partiallysaturated or fully unsaturated. That is, hydrogens attached to any ofthe carbons at positions 1-17 may be omitted so as to allow unsaturationwithin the A, B, C and/or D ring. For example, when carbons at numerals5, 8, 9 and 14 are indicated as being substituted with one hydrogen, andit is also indicated that each of rings A, B, C and D is independentlyfully saturated, partially saturated or fully unsaturated, then any oneor more of the hydrogens attached to carbons at numerals 5, 8, 9 and 14may be omitted in order to allow unsaturation at the carbon atom.

[0106] The compounds of the present invention are intended aspharmaceutical agents. Preferably, the molecular weight of a compound ofthe invention is relatively small, that is, less than about 5,000 g/mol,typically less than 4,000 g/mol, more typically less than 3,000 g/mol,still more typically less than 2,000 g/mol, yet more typically less than1,000 g/mol, where the minimum molecular weight of a compound of theinvention is about 300 g/mol, and each of these typical ranges is aseparate embodiment of the present invention.

[0107] The steroid compounds of the present invention includepharmaceutically acceptable salts, solvates, stereoisomers and prodrugsof the 3-nitrogen-6,7-dioxygenated steroid structures described above,in isolation or in mixtures with one another.

[0108] The steroid compounds of the invention may, and typically do,exist as solids, including crystalline solids which can be crystallizedfrom common solvents such as ethanol, N,N-dimethyl-formamide, water, orthe like. The crystallization process may, depending on thecrystallization conditions, provide various polymorphic structures.Typically, a more thermodynamically stable polymorph is advantageous tothe commercial scale manufacture of a steroid compound of the invention,and is a preferred form of the compound.

[0109] Often, crystallizations produce a solvate of the steroid compoundhaving the structure shown above. As used herein, the term “solvate”refers to an aggregate that comprises one or more3-nitrogen-6,7-dioxygenated steroid compounds of the invention, with oneor more molecules of solvent. The solvent may be water, in which casethe solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The steroid compounds may be truesolvates, while in other cases, the steroid may merely retainadventitious water or be a mixture of water plus some adventitioussolvent.

[0110] As used herein, a “pharmaceutically acceptable solvate” refers toa solvate that retains the biological effectiveness and properties ofthe biologically active 3-nitrogen-6,7-dioxygenated steroid compounds ofthe invention. Examples of pharmaceutically acceptable solvates include,but are not limited to, water, isopropanol, ethanol, methanol, DMSO,ethyl acetate, acetic acid, and ethanolamine. It should be appreciatedby those skilled in the art that solvated forms are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention. Sykes, P. A., Guidebook to Mechanism in OrganicChemistry, 6th Ed (1986, John Wiley & Sons, N.Y.) is an exemplaryreference that describe solvates.

[0111] The inventive compounds may exist as single stereoisomers,racemates and/or mixtures of enantiomers and/or diastereomers. All suchsingle stereoisomers, racemates and mixtures thereof are intended to bewithin the scope of the present invention. In a preferred aspect, theinventive compounds are used in optically pure form.

[0112] A “pharmaceutically acceptable prodrug” is intended to mean acompound that may be converted under physiological conditions or bysolvolysis to a biologically active 3-nitrogen-6,7-dioxygenated steroidcompound as described above. Thus, the term “prodrug” refers to ametabolic precursor of a steroid compound of the present invention thatis pharmaceutically acceptable. A prodrug may be inactive whenadministered to a subject but is converted in vivo to an active3-nitrogen-6,7-dioxygenated steroid compound of the invention. Prodrugsare typically rapidly transformed in vivo to yield the parent compoundof the above formulae, for example, by hydrolysis in blood.

[0113] A discussion of prodrugs is provided in T. Higuchi and V. Stella,“Pro-drugs as Novel Delivery Systems,” Vol 14 of the A. C. S. SymposiumSeries, and in Bioreversible Carriers in Drug Design, ed. Edward B.Roche, American Pharmaceutical Association and Pergamon Press, 1987,both of which are incorporated herein by reference. A typical prodrug isa derivative of the steroid compounds of the invention which havechemically or metabolically cleavable groups and become, by solvolysisor under physiological conditions, the compounds of the invention whichare pharmaceutically active in vivo. The prodrug derivative form oftenoffers advantages of solubility, tissue compatibility, or delayedrelease in a mammalian organism (see, Bundgard, H., Design of Prodrugs,pp. 7-9, 21-24, Elsevier, Amsterdam 1985). A preferred prodrug is acompound having substitution at the 3-nitrogen atom of the steroids ofthe invention, where the substitution is cleaved in vivo to provide apharmaceutically active compound.

[0114] Steroids of the present invention having C3 nitrogen substitutionand oxygen substitution at positions 6 and 7 have unexpected propertiesthat enhance the efficacies of these compounds. For instance, thesteroid of the present invention have an excellent metabolic stabilityin S9 fractions from human liver. For example, 100% of compounds 28, 89,139, and 143 remains unchanged after 15 and even 30 minutes incubationwith human S9 fractions. It was an unexpected finding that C3 nitrogensubstitutions significantly decrease glucuronidation of the molecules inplasma. In addition, steroids of the present invention having C3nitrogen substitutions such as compounds 28, 89 and 83 are highlysoluble in aqueous solution, demonstrating a solubility of >100 mg/ml inwater. Furthermore, the potency and pharmacokinetic profile of steroidsof the present invention with C3 nitrogen substitutions is highlysuitable for therapeutic application. Doses of <1.0 mg/kg once per dayreproducibly demonstrate significant anti-inflammatory activity in invivo inflammation models. In the rat, compounds 28 and 89 have anaverage half-life of 7.5 hours and an oral bioavailability of ˜100%,while in the monkey, the half-life averages 15 hours and oralbioavailability is 25-30%. The maximal concentration in plasma in bothspecies is predictable and linear.

[0115] C. Preparation of Compounds

[0116] The compounds according to the present invention can be preparedby methods employing steps known to those skilled in the art oranalogous to those steps. General methods for the reactions on steroidscan be found in “Steroid Reactions”, C. Djerassi, Ed. Holden Day, SanFrancisco, Calif., 1963 and references cited therein. General syntheticmethods can be found in “Comprehensive Organic Transformations”, R. C.Larock, VCH Publishers, New York, N.Y., 1989 and references citedtherein. Additional literature references useful for the synthesis ofcompounds of the invention are as follows: T. Reichstein; C. H. Meystre,Helv. Chim. Acta, 1932, 22, 728; H. Westmijze; H. Kleyn; P. Vermeer; L.A. van Dijok, Tet. Lett. 1980, 21, 2665; K. Prezewowsky; R Wiechert,U.S. Pat. No. 3,682,983; P. Kaspar; H. Witzel, J. Steroid Biochem. 1985,23, 259; W. G. Dauben; T. Brookhart, J. Am. Chem. Soc. 1981, 103, 237;A. J. Manson et al., J. Med. Chem. 1963, 6, 1; R. O. Clinton et al, J.Am. Chem. Soc. 1961, 83, 1478; and J. A. Zderic et al. Chem. and Ind.1960, 1625.

[0117] In a preferred method, C3, C6, C7 and C17 polyoxygenated steroidsare used as starting materials or intermediates. Methods to introduce C6and C7 oxygens into commercially available starting materials aredescribed in U.S. Pat. No. 6,046,185. This U.S. Patent also disclosesmany ways in which substitution and preferred stereochemistry can beintroduced into positions C1, C2, C4, C5, C8, C9, C10, C11, C12, C13,C14, C15, C16 and C17. In the present invention, the C6 and C7 oxygensmay be present as hydroxyls or as protected hydroxyls. The 6- and7-hydroxyls can be protected individually or they can together be partof a ring. Suitable protecting groups are listed in Greene and Wuts,“rotective Groups in Organic Synthesis”, John Wiley & Sons, New York,N.Y. (1999).

[0118] Referring to Scheme A, ketones of compound 2, or compoundsanalogous thereto, can be alkylated with a variety of alkylating groupsto give steroids of the invention having but not limited to, alkyl,cycloalkyl, aryl, and heteroaryl substitution. For example, alkylationof the 17-ketone 2, with the anion of acetylene generates the17α-ethynyl-17β-hydroxyl intermediate 3. Reversal of the stereochemistryof the C17 substituents may be carried out by first forming themethylsulfonate followed by treatment with silver (I) nitrate intetrahydrofuran (THF) and water. Dehydration of compound 3 using POCl₃in 2,4lutidine gives compound 4. Tetrabutylammonium fluoride in THFremoves the tert-butyldinethylsilyl (TBS) protecting group from the3-hydroxyl to give compound 5. Treatment of the 3α-hydroxyl compound 5with ZnN₆.2py, triphenylphosphine and diisopropyl azodicarboxylate(DIAD) in toluene gives the 3β-azido compound 6. The ZnN₆.2py isprepared by the reaction of Zn(NO₃)₂ and NaN₃ followed by treatment withpyridine according to the procedure of M. C. Viaud and P. Rollin inSyntdlesis 1990, 130. Lithium aluminum hydride reduction of the azide indiethyl ether (Et₂O) gives the amine 7. Treatment with HCl in THF andwater removes the acetonide group and forms the ammonium chloride salt8.

[0119] Referring to Scheme B, steroids of the invention having allenefunctionality may be prepared from intermediates analogous to compound3. Exemplary is the reaction of compound 3 with LiAlH₄ and AlCl₃ in THFto give the allene 9. Tetrabutylammonium fluoride in THF removes theprotecting group from the 3-hydroxyl to give compound 10. Treatment ofthe 3α-hydroxyl compound 10 with ZnN₆.2py, triphenylphosphine and DIADin toluene gives the 3β-azido compound 11. Lithium aluminum hydridereduction of the azide 11 in Et₂O gives the amine 12. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 13.

[0120] Referring to Scheme C, compounds of the invention having alkynylfunctionality may be prepared from allene intermediates. Exemplary isthe treatment of compound 9 with n-BuLi in THF giving the 17β-ethynylcompound 14. Tetrabutylammonium fluoride in THF removes the protectinggroup from the 3-hydroxyl to give compound 15. Treatment of the3α-hydroxyl compound 15 with ZnN₆.2py, triphenylphosphine and DIAD intoluene gives the 3β-azido compound 16. Lithium aluminum hydridereduction of the azide 16 in Et₂O gives the amine 17. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 18.

[0121] Referring to Scheme D, steroids of the invention having alkenylfunctionality may be prepared from alkyne intermediates. Exemplary isthe controlled hydrogenation of compound 14 using Pd-CaCO₃ as catalystto give the alkene 19. Tetrabutylammonium fluoride in THF removes theprotecting group from the 3-hydroxyl to give compound 20. Treatment ofthe 3α-hydroxyl compound 20 with ZnN₆.2py, triphenylphosphine and DIADin toluene gives the 3β-azido compound 21. Lithium aluminum hydridereduction of the azide 21 in Et₂O gives the amine 22. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 23.

[0122] Compound 2 can be used in a multitude of olefination reactions,including Wittig-type reactions to provide compounds of the inventionhaving an exocyclic olefin at C17. For example, as illustrated in SchemeE, compound 2 may be treated with ethyltriphenylphosphonium bromide andpotassium tert-butoxide (KO^(t)Bu) to provide compound 24 havingR₁=methyl and R₂=hydrogen. Tetrabutylammonium fluoride in THF removesthe protecting group from the 3-hydroxyl to give compound 25. Treatmentof the 3α-hydroxyl compound 25 with ZnN₆.2py, triphenylphosphine andDIAD in toluene gives the 3β-azido compound 26. Lithium aluminum hydridereduction of the azide 26 in Et₂O gives the amine 27. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 28 having R¹=methyl and R₂=hydrogen.

[0123] In analogy to the synthesis shown in Scheme E, ketones such ascompound 2 may be reacted with other Wittig-type reagents such as, butnot limited to, methyl-, propyl-, butyl-, pentyl-, orhexyltriphenyiphosphonium bromide to give steroids of the inventionanalogous to compound 28 having R₂=hydrogen and R¹=hydrogen, ethyl,propyl, butyl or pentyl.

[0124] Steroids of the invention can contain exocyclic double bonds of Eand/or Z geometry. For example, as illustrated in Scheme F, the Z-olefin24 in cyclohexane may be treated with UV light in the presence ofdiphenyldisulfide resulting in isomerization to the E-olefin 29.Tetrabutylammonium fluoride in THF removes the protecting group from the3-hydroxyl to give compound 30. Treatment of the 3α-hydroxyl compound 30with ZnN₆.2py, triphenylphosphine and DIAD in toluene gives the 3β-azidocompound 31. Lithium aluminum hydride reduction of the azide 31 in Et₂Ogives the amine 32. Treatment with HCl in THF and water removes theacetonide group and forms the ammonium chloride salt 33.

[0125] A multitude of steroids of the invention having functionalizedsidechains can be prepared using methods such as Lewis acid promotedcouplings to alkehydes and Michael acceptors. For example, asillustrated in Scheme G, compound 24 may be reacted with methylpropiolate in the presence of diethylaluminum chloride to give compound34. The double bonds may be hydrogenated using a catalyst such asplatinum to give compound 35. Tetrabutylammonium fluoride in THF removesthe protecting group from the 3-hydroxyl to give compound 36. Treatmentof the 3α-hydroxyl compound 36 with ZnN₆.2py, triphenylphosphine andDIAD in toluene gives the 3β-azido compound 37. Hydrogenation of theazide 37 using a palladium catalyst gives the amine 38. Treatment withHCl in THF and water removes the acetonide group and forms the ammoniumchloride salt 39.

[0126] With a 3-amino steroid, such as prepared by any of the aboveSchemes A-G, a large variety of secondary and tertiary amine compoundsof the invention can be prepared. FIGS. 1A and 1B outline severalsynthetic pathways that may be employed to prepare 3-amino compounds ofthe present invention. For instance, reductive amination methods may beused to couple primary (see FIG. 1A) and secondary (see FIG. 1B) amineswith aldehydes (RC(═O)H) and ketones RC(═O)R′). Although not shown ineither of FIGS. 1A or 1B, compounds having two aldehyde groups, i.e.,dialdehydes of the general formula HC(═O)—R—C(═O)H, may be reacted with3-amino steroids to provide steroids having heterocyclic structures atthe 3-position. In addition (or alternatively), reductive aminationmethods may be used to couple 3-keto steroids with heterocyclicsecondary amines. By these approaches, the present invention providescompounds wherein R¹ and R² may, together with the N to which they areboth bonded, form a heterocyclic structure that may be part of anorganic group having 1-30 carbons and optionally containing 1-6heteroatoms selected from nitrogen, oxygen and silicon. Commercialsources and reference to the chemical literature provides one ofordinary skill in the art with access to a multitude of aldehydes(including dialdehyes) and ketones that may be used to prepare steroidcompounds of the present invention. Reductive amination methods aredescribed in, for example Synthesis 1975, 135; J. Am. Chem. Soc. 1971,93, 2897; M. Freifelder in “Catalytic Hydrogenation in OrganicSynthesis” J. Wiley & Sons 1978, Ch. 10; Russ. Chem. Rev. 1980, 49, 14,and references cited therein. See also, J. Chem. Soc. Perkin Trans 11998, 2527; and Synlett 1999, 1781, as well as references cited therein.

[0127] Primary (see FIG. 1A) and secondary (see FIG. 1B) amines can becoupled to aryl compounds (ArX) to generate a variety of arylsubstituted amine compounds of the invention. Commercial sources andreference to the chemical literature provides one of ordinary skill inthe art with access to a multitude of aryl compounds that may be used toprepare steroid compounds of the present invention. Examples of methodsfor the amination of aryl compounds can be found in J. Org. Chem. 2000,65, 1158 and in the review Angew. Chem. Int. Ed. 1998, 37, 2046 andreferences cited therein.

[0128] Methods to react primary (see FIG. 1A) and secondary (see FIG.1B) amines with acyl chlorides (RC(═O)Cl) and sulfonyl chlorides(RSO₂Cl) to generate amide and sulfonamide compounds of the invention,respectively, are well known to those skilled in the art of organicchemistry, in the context of other amine compounds, and these sametechniques may be applied to the amine compounds of the presentinvention. Commercial sources and reference to the chemical literatureprovides one of ordinary skill in the art with access to a multitude ofacyl chlorides and sulfonyl chlorides that may be used to preparesteroid compounds of the present invention.

[0129] Methods to react primary (see FIG. 1A) and secondary (see FIG.1B) amines with isocyanates (RN═C═O) and isothiocyanates (RN═C═S) togenerate ureas and thioureas, respectively, are also well known to thoseskilled in the art of organic chemistry, in the context of other aminecompounds, and these same techniques may be applied to the aminecompounds of the present invention. Commercial sources and reference tothe chemical literature provides one of ordinary skill in the art withaccess to a multitude of isocyanates and isothiocyanates that may beused to prepare steroid compounds of the present invention. The reviewarticle Russ. Chem. Rev. 1985, 54, 249 and references cited thereindescribes examples of the variety of substituted ureas and thioureasthat can be encompassed by the invention.

[0130] Thus, by using appropriately selected aldehydes, ketones, arylcompounds, acyl chlorides, sulfonyl chlorides, isocyanates and/orisothiocyanates, one of ordinary skill in the art may prepare steroidcompounds wherein R¹ and R² are selected from hydrogen and organicgroups having 1-30 carbons and optionally containing 1-6 heteroatomsselected from nitrogen, oxygen, phosphorous, silicon, and sulfur.

[0131] Steroids of the invention may have fused heterocycles such as,but not limited to, pyrazole, isoxazole and pyrimidine. As illustratedin Scheme H, compound 43 is an example of a fused pyrazole of theinvention, for which the synthesis of the starting material compound 40is described in U.S. Pat. No. 6,046,185. Treatment of compound 40 withethyl formate in pyridine in the presence of NaOMe gives thehydroxymethylene intermediate 41. Reaction of compound 41 with hydrazinehydrate in EtOH forms the pyrazole compound 42, which upon treatmentwith tetrabutylammonium fluoride in THF gives the compound 43.

[0132] As illustrated in Scheme I, intermediates such as compound 41 maybe converted into isoxazoles of which compound 44 is exemplary.Treatment of the hydroxymethylene intermediate 41 with ammoniumhydroxide in pyridine followed by deprotection of the 6- and 7-hydroxylsusing tetrabutylammonium fluoride in THF gives the isoxazole 44.

[0133] As illustrated in Scheme J, intermediates such as compound 41 maybe converted into pyrimidines of which compound 44a is exemplary.Treatment of the hydroxymethylene intermediate 41 with benzamidinehydrochloride and potassium hydroxide in ethanol followed bydeprotection of the 6- and 7-hydroxyls using tetrabutylammonium fluoridein THF gives the pyrimidine 44a.

[0134] Thus, by proceeding through compounds having keto substitution ascarbon 3 and ═CHOH substitution at carbon 2, the present inventionprovides access to a multitude of compounds wherein R¹ may be a 2 or 3atom chain to numeral 2 so that —N—R¹— forms part of a fused bicyclicstructure to ring A.

[0135] Reaction of 3-keto steroids with hydroxylamine and pyridine maybe employed to produce steroid oximes of the invention. A steroid oximehas R² as a direct bond to numeral 3, thus providing a double bondbetween the carbon at numeral 3 and the N, and R¹ is OH. Primary aminesmay be oxidized to nitro compounds by, for instance, dimethyldioxirane.Thus, R¹ and R² may be oxygen. Methods to generate the nitrofunctionality are described in J. Org. Chem. 1989, 54, 5783. Reaction of3-ketones with dimethylhydrazine gives N,N-dimethylhydrazone steroids ofthe invention in which R² is a direct bond to numeral 3 and R¹ is NMe₂.Treatment of dimethylhydrazone steroids with hydrazine generateshydrazone steroids of the invention. Description of the methods togenerate N,N-dimethylhydrazones and hydrazones can be found in J. Org.Chem. 1966, 31, 677. Primary amines may also be reacted by methodologyknown in the art with sulfonic/sulfuric acids and esters to providesulfamate compounds, i.e., steroids wherein numeral 3 is bonded to—N—SO₃—R and R is H or an organic group having 1-30 carbons andoptionally containing 1-6 heteroatoms selected from nigrogen, oxygen,phosphorous, silicon, and sur.

[0136] D. Pharmaceutical Compositions

[0137] The present invention provides a pharmaceutical or veterinarycomposition (hereinafter, simply referred to as a pharmaceuticalcomposition) containing a compound of formula (1) as described above, inadmixture with a pharmaceutically acceptable carrier. The inventionfurther provides a composition, preferably a pharmaceutical composition,containing an effective amount of a compound as described above, inassociation with a pharmaceutically acceptable carrier.

[0138] The pharmaceutical compositions of the present invention may bein any form which allows for the composition to be administered to apatient. For example, the composition may be in the form of a solid,liquid or gas (aerosol). Typical routes of administration include,without limitation, oral, topical, parenteral, sublingual, rectal,vaginal, ocular, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrastemal injection or infusion techniques. Pharmaceutical compositionof the invention are formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a patient. Compositions that will be administered to apatient take the form of one or more dosage units, where for example, atablet may be a single dosage unit, and a container of a compound offormula (1) in aerosol form may hold a plurality of dosage units.

[0139] Materials used in preparing the pharmaceutical compositionsshould be pharmaceutically pure and non-toxic in the amounts used. Itwill be evident to those of ordinary skill in the art that the optimaldosage of the active ingredient(s) in the pharmaceutical compositionwill depend on a variety of factors. Relevant factors include, withoutlimitation, the type of subject (e.g., human), the particular form ofthe active ingredient, the manner of administration and the compositionemployed.

[0140] In general, the pharmaceutical composition includes an (where “a”and “an” refers here, and throughout this specification, as one or more)active compound of formula (1) as described herein, in admixture withone or more carriers. The carrier(s) may be particulate, so that thecompositions are, for example, in tablet or powder form. The carrier(s)may be liquid, with the compositions being, for example, an oral syrupor injectable liquid. In addition, the carrier(s) may be gaseous, so asto provide an aerosol composition useful in, e.g., inhalatoryadministration.

[0141] When intended for oral administration, the composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

[0142] As a solid composition for oral administration, the compositionmay be formulated into a powder, granule, compressed tablet, pill,capsule, chewing gum, wafer or the like form. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following adjuvants may be present: binderssuch as carboxymethylcellulose, ethyl cellulose, microcrystallinecellulose, or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin, a flavoring agent such as peppermint,methyl salicylate or orange flavoring, and a coloring agent.

[0143] When the composition is in the form of a capsule, e.g. a gelatincapsule, it may contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0144] The composition may be in the form of a liquid, e.g., an elixir,syrup, solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

[0145] The liquid pharmaceutical compositions of the invention, whetherthey be solutions, suspensions or other like form, may include one ormore of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, cyclodextrin, propylene glycol or othersolvents; antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

[0146] A liquid composition intended for either parenteral or oraladministration should contain an amount of a compound of formula (1)such that a suitable dosage will be obtained. Typically, this amount isat least 0.01% of a compound of the invention in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1% and about 80% of the weight of the composition. Preferredoral compositions contain between about 4% and about 50% of the activecompound of formula (1). Preferred compositions and preparationsaccording to the present invention are prepared so that a parenteraldosage unit contains between 0.01% to 2% by weight of active compound.

[0147] The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of formula (1) of from about 0.1% to about10% w/v (weight per unit volume).

[0148] The composition may be intended for rectal administration, in theform, e.g., of a suppository which will melt in the rectum and releasethe drug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

[0149] The composition may include various materials which modify thephysical form of a solid or liquid dosage unit. For example, thecomposition may include materials that form a coating shell around theactive ingredients. The materials which form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule.

[0150] The composition in solid or liquid form may include an agentwhich binds to the active component(s) and thereby assists in thedelivery of the active components. Suitable agents which may act in thiscapacity include a monoclonal or polyclonal antibody, a protein or aliposome.

[0151] The pharmaceutical composition of the present invention mayconsist of gaseous dosage units, e.g., it may be in the form of anaerosol. The term aerosol is used to denote a variety of systems rangingfrom those of colloidal nature to systems consisting of pressurizedpackages. Delivery may be by a liquefied or compressed gas or by asuitable pump system which dispenses the active ingredients. Aerosolsof, compounds of the invention may be delivered in single phase,bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, spacers and the like, which togethermay form a kit. Preferred aerosols may be determined by one skilled inthe art, without undue experimentation.

[0152] Whether in solid, liquid or gaseous form, the pharmaceuticalcomposition of the present invention may contain one or more knownpharmacological agents used in the treatment of inflammation (includingasthma, allergy, rheumatoid arthritis, multiple sclerosis, etc.),proliferative disorders (cancers), diseases treatable through theregulation of calcium (including hypertension, cardiac arrhythmias,etc.), and Acquired Immune Deficiency Syndrome (AIDS).

[0153] The pharmaceutical compositions may be prepared by methodologywell known in the pharmaceutical art.

[0154] A composition intended to be administered by injection can beprepared by combining the compound of formula (1) with water so as toform a solution. A surfactant may be added to facilitate the formationof a homogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of formula (1) so as tofacilitate dissolution or homogeneous suspension of the active compoundin the aqueous delivery system.

[0155] E. Biological Activity

[0156] The compounds disclosed herein of formula 1, or compositionscomprising one of more of these compounds and a pharmaceuticallyacceptable carrier, diluent or excipient, may be used in a method fortreating or preventing an inflammatory condition or disease in apatient, where the method comprises administering to the patient in needthereof an amount of a compound or composition according to the presentinvention, where the amount is effective to treat or prevent theinflammatory condition or disease of the patient.

[0157] The inflammatory condition or disease may involve respiratoryinflammation (e.g., wherein the respiratory disease is asthma, orwherein the respiratory disease is chronic obstructive pulmonarydisease; or wherein the respiratory disease is emphysema); theinflammatory condition may be an autoimmune condition or disease: theinflammatory condition or disease may be lupus erythematosus disease;the inflammatory condition or disease may involve acute or chronicinflammation of bone and/or cartilage compartments of joints; theinflammatory condition or disease may be an arthritis selected fromrheumatoid arthritis, gouty arthritis or juvenile rheumatoid arthritis;the inflammatory condition or disease may be a central nervous systemdisease; the condition or disease may be associated with leukocyteinfiltration; the condition or disease may be associated with edema; thecondition or disease may be associated with ischemia reperfusion injury;the condition or disease may be associated with elevated levels ofinflammatory cytolines (e.g., wherein the inflammatory cytokine isinterleukin (IL)-4, or wherein the inflammatory cytokine is IL-5, orwherein the inflammatory cytoldne is IL-10, or wherein the inflammatorycytokine is IL-13, or wherein the inflammatory cytokine is IL-9, orwherein the inflammatory cytokine is IL-1, or wherein the inflammatorycytokine is IL-2, or wherein the inflammatory cytokine is IL-6, orwherein the inflammatory cytokine is IL-18, or wherein the inflammatorycytokine is IL-3, or wherein the inflammatory cytokine is IL-8, orwherein the inflammatory cytokine is IL-12, or wherein the inflammatorycytokine is TNF-α, or wherein the inflammatory cytokine is TGF-β, orwherein the inflammatory cytoline is GM-CSF, or wherein the inflammatorycytokine is IFN-γ, or wherein the inflammatory cytokine is LTB4, orwherein the inflammatory cytokine is a member of the cysteinylleukotriene family, or wherein the inflammatory cytokine is regulated onactivation normal T cell expressed and secreted (RANTES), or wherein theinflammatory cytokine is eotaxin-1, 2, or 3, or wherein the inflammatorycytokine is macrophage inflammatory protein (MIP)-1α, or wherein theinflammatory cytokine is monocyte chemoattractant protein-1, 2, 3, or4,); the condition or disease may be associated with altered levels ofinflammatory adhesion molecules (e.g., wherein the adhesion molecule isvascular cell adhesion molecule (VCAM-1 or 2), wherein the adhesionmolecule is intercellular adhesion molecule (ICAM-1 or 2), wherein theadhesion molecule is very late antigen-4 (VLA-4), wherein the adhesionmolecule is leukocyte function associated antigen-1 (LFA-1); wherein theadhesion molecule is a selectin); the inflammatory condition or diseasemay be multiple sclerosis; the inflammatory condition or disease may bepulmonary sarcadosis; the inflammatory condition or disease may beocular inflammation or allergy; the inflammatory condition or diseasemay be allergic rhinitis; the inflammatory condition or disease may bean inflammatory bowel disease (e.g., Crohn's disease or ulcerativecolitis); the inflammatory condition or disease may be an inflammatorycutaneous disease (e.g., psoriasis or dermatitis); the inflammatorycondition or disease may be graft vs host disease; the inflammatorycondition or disease may be vascular (e.g., vasculitis); theinflammatory condition or disease may be an atherosclerotic disease.

[0158] Furthermore, the present invention provides a method for treatingor preventing a disease or condition in a patient, where the disease orcondition is associated with pathological conditions that involveleukocyte infiltration, the method comprising administering to a patientin need thereof an amount of a compound or a composition of the presentinvention, wherein the amount is effective to treat or prevent a diseaseor condition associated with pathological conditions that involveleukocyte infiltration.

[0159] Furthermore, the present invention provides a method of treatingor preventing asthma in a patient, comprising administering to a patientin need thereof an amount of a compound or composition of the presentinvention, where the amount is effective to treat or prevent asthma inthe patient.

[0160] Furthermore, the present invention provides a method of treatingor preventing allergy in a patient, comprising administering to apatient in need thereof an amount of a compound or composition of thepresent invention, where the amount is effective to treat or preventallergy in the patient.

[0161] In a method of the present invention, a compound of formula (1);or a composition comprising one or more compounds of formula (1) and apharmaceutically acceptable carrier, diluent or excipient, may, althoughneed not, achieve one or more of the following desired results in thesubject to whom has been administered a compound of formula (1) asdefined above, or a composition containing one of these compounds and apharmaceutically acceptable carrier, diluent or excipient:

[0162] 1. Inhibition of leukocyte infiltration (e.g., neutrophils,eosinophils, etc.)

[0163] 2. Inhibition of leukocyte activation

[0164] 3. Alteration of lymphocyte ratio (e.g., TH1 vs TH2 cells)

[0165] 4. Inhibition of leukocyte chemotaxis;

[0166] 5. inhibition of TNF-α production and/or release;

[0167] 6. Inhibition of chemokine production and/or release (e.g.,eotanin, etc.);

[0168] 7. Inhibition of adhesion molecule production, release and/orfunction (e.g., VCAM, VLA-4, etc.);

[0169] 8. Inhibition of edema;

[0170] 9. Inhibition of interleukin cytokine production and/or release(e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-8, IL-9, IL10, IL-12, IL-13,IL-18,);

[0171] 10. Inhibition of inflammatory mediator release (e.g.,leukotrienes, tryptase, adenosine etc.);

[0172] 11. Inhibition of histamine release;

[0173] 12. Inhibition of parameters of asthma; and

[0174] 13. Inhibition of parameters of allergy.

[0175] The compounds disclosed herein of formula 1 (i.e., compounds offormulae (1), or compounds of the present invention), or compositionscomprising one of more of these compounds and a pharmaceuticallyacceptable carrier, diluent or excipient, may be used in a method fortreating or preventing a proliferative disorder in a patient, where themethod comprises administering to the patient in need thereof an amountof a compound or composition according to the present invention, wherethe amount is effective to treat or prevent the proliferative disorderof the patient. As used herein, proliferative disorders includes,without limitation, all leukemias and solid tumors that are susceptibleto undergoing differentiation or apoptosis upon interruption of theircell cycle.

[0176] The compounds disclosed herein of formula 1 (i.e., compounds offormulae (1), or compounds of the present invention), or compositionscomprising one or more of these compounds and a pharmaceuticallyacceptable carrier, diluent or excipient, may be used in a method fortreating or preventing diseases treatable through regulation of calciumin a patient, where the method comprises administering to the patient inneed thereof an amount of a compound or composition according to thepresent invention, where the amount is effective to treat or prevent thedisease of the patient. As used herein, diseases treatable throughregulation of calcium includes, without limitation, cardiac arrhythmia,atrial fibrillation, acute coronary syndromes, hypertension, ischemiareperfusion injury, stroke, epilepsy, demyelinating diseases such asmultiple sclerosis, pain, status epilepticus, artherosclerosis, anddiabetes.

[0177] The compounds disclosed herein of formula 1 (i.e., compounds offormulae (1), or compounds of the present invention), or compositionscomprising one or more of these compounds and a pharmaceuticallyacceptable carrier, diluent or excipient, may be used in a method fortreating or preventing Acquired Immunodeficiency Syndromes (AIDS) in apatient, where the method comprises administering to the patient in needthereof an amount of a compound or composition according to the presentinvention, where the amount is effective to treat or prevent theAcquired Immunodeficiency Syndromes of the patient. As used herein,Acquired Immunodeficiency Syndromes through infection with humanimmunodeficiency virus type 1 includes, without limitation, associatedcomplications such as Acquired Immunodeficiency Syndrome DementiaComplex, and neuro-Acquired Immunodeficiency Syndromes.

[0178] Thus, the inventive method may be used to treat inflammation,including both acute and chronic inflammation, as well as certainproliferative disorders (cancers), diseases treatable through regulationof calcium, and AIDS. As used herein, inflammation includes, withoutlimitation, ankylosing spondylitis, arthritis (where this termencompasses over 100 kinds of rheumatic diseases), asthma, chronicobstructive pulmonary disease, allergy, allergic rhinitis, Crohn'sdisease, fibromyalgia syndrome, gout, inflammations of the brain(including multiple sclerosis, AIDS dementia, Lyme encephalopathy,herpes encephalitis, Creutzfeld-Jakob disease, and cerebraltoxoplasmosis), emphysema, inflammatory bowel disease, irritable bowelsyndrome, ischemia-reperfusion injury, atopic dermatitis, juvenileerythematosus pulmonary sarcoidosis, Kawasaki disease, osteoarthritis,pelvic inflammatory disease, psoriatic arthritis (psoriasis), rheumatoidarthritis, psoriasis, tissue/organ transplant, graft vs host disease;scleroderma, spondyloarthropathies, systemic lupus erythematosus,pulmonary sarcoidosis, vasculitis, artherosclerosis, cardiomyopathy,autoimmune myocarditis, and ulcerative colitis.

[0179] The inventive method provides for administering a therapeuticallyeffective amount of a compound of formula (1), including salts,compositions etc. thereof As used herein, the actual amount encompassedby the term “therapeutically effective amount” will depend on the routeof administration, the type of warm-blooded animal being treated, andthe physical characteristics of the specific warm-blooded animal underconsideration. These factors and their relationship to determining thisamount are well known to skilled practitioners in the medical arts. Thisamount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors that those skilled in themedical arts will recognize.

[0180] An effective amount of a compound or composition of the presentinvention will be sufficient to treat inflammation, proliferativediseases, diseases treatable by regulation of calcium, or AIDS, in awarm-blooded animal, such as a human. Methods of administering effectiveamounts of anti-inflammatory agents are well known in the art andinclude the administration of inhalation, oral or parenteral forms. Suchdosage forms include, but are not limited to, parenteral solutions,tablets, capsules, sustained release implants and transdermal deliverysystems; or inhalation dosage systems employing dry powder inhalers orpressurized multi-dose inhalation devices.

[0181] The dosage amount and frequency are selected to create aneffective level of the agent without harmful effects. It will generallyrange from a dosage of about 0.001 to 100 mg/Kg/day, and typically fromabout 0.01 to 10 mg/Kg/day where administered orally or intravenously.Also, the dosage range will be typically from about 0.0001 to 10mg/Kg/day where administered intranasally or by inhalation.

[0182] The compounds of formula (1) including the compounds used in themethods and compositions set forth above, may be prepared according tothe Schemes set forth in the following examples. The following examplesare offered by way of illustration and not by way of limitation.

[0183] Unless otherwise stated, flash chromatography and columnchromatography may be accomplished using Merck silica gel 60 (230-400mesh). Flash chromatography may be carried out according to theprocedure set forth in: “Purification of Laboratory Chemicals”, 3rd.edition, Butterworth-Heinemann Ltd., Oxford (1988), Eds. D. D. Perrinand W. L. F. Armarego, page 23. Column chromatography refers to theprocess whereby the flow rate of eluent through a pacling material isdetermined by gravity. In all cases flash chromatography and radialchromatography may be used interchangeably. Radial chromatography isperformed using silica gel on a Chromatotron Model #7924T (HarrisonResearch, Palo Alto, Calif.). Unless otherwise stated, quoted R_(f)values are obtained by thin layer chromatography using Silica Gel 60F₂₅₄ (Merck KGaA, 64271, Darmstadt, Germany). Brine refers to asaturated solution of sodium chloride.

[0184] Also, unless otherwise stated, chemical reactants and reagentswere obtained from standard chemical supply houses, such as Aldrich(Milwaukee, Wis.; www.aldrich.sial.com); EM Industries, Inc. (Hawthorne,N.Y.; www.emscience.com); Fisher Scientific Co. (Hampton, N.H.;www.fischerl.com); and Lancaster Synthesis, Inc. (Windham, N.H.;www.lancaster.co.uk). Sulfo-NHS-biotin was obtained from Pierce(Rockford, Ill., www.piercenet.com). MP-TsOH resin, PS-DIEA resin,PS-Trisamine resin and PS-Benzaldehyde resin were obtained from ArgonautTechnologies (San Carlos, Calif., www.argotech.com). Gases were obtainedfrom Praxair (Vancouver, B.C.). Cell lines, unless otherwise stated,where obtained from public or commercial sources, e.g., American TissueCulture Collection (ATCC, Rockville, Md.).

SYNTHESIS EXAMPLES Example 1 3-Amino-6,7-dihydroxy-17-ethylidene Steroid

[0185] Compound 49, a representative compound of the invention, isprepared according to Scheme 1. Any number of compounds related tocompound 49 could be produced using similar methodology. The startingmaterial compound 45 can be prepared according to the methodologydescribed in U.S. Pat. No. 6,046,185. Olefination of the ketone 45 isaccomplished using ethyltriphenylphosphonium bromide and KO^(t)Bu intoluene. Treatment of the 3β-hydroxyl compound 46 with ZnN₆.2py,triphenylphosphine and DIAD in toluene produced the 3α-azido compound47. Lithium aluminum hydride reduction of the azide in Et₂O provided theamine 48. Treatment with HCl in THF and water removes the acetonidegroup and forms the ammonium chloride salt 49.

[0186] Synthesis of Compound 46

[0187] A solution of KO^(t)Bu (0.24 g, 2.0 mmol), EtPPh₃Br (0.75 g, 2.0mmol) and toluene (2.5 ml) was stirred at room temperature under argon.After 1 hour the deep red solution was cooled in ice and the ketone 45(184 mg, 0.508 mmol) was added and the resulting solution was allowed towarm to room temperature. After stirring overnight the reaction wasquenched with 10 ml of water, diluted with 60 ml of ethyl acetate(EtOAc), separated and washed with 2×10 ml of brine, dried over MgSO₄,filtered and concentrated. Purification by column chromatography elutingwith 1:1 EtOAc/hexanes afforded 171 mg (90%) of compound 46 as acolorless film.

[0188] Synthesis of Compound 47

[0189] DIAD (0.44 ml, 2.14 mmol) was added dropwise over 10 minutes to aroom temperature solution of the 3β-hydroxy compound 46 (400 mg, 1.07mmol), ZnN₆.2py (246 mg, 0.80 mmol), Ph₃P (560 mg, 2.14 mmol) andtoluene (10.7 ml) under argon. After 4 hours the reaction mixture wasloaded onto a column of silica gel packed in 10% ethyl acetate/hexanesand eluted with 20% ethyl acetate/hexanes to afford 422 mg (99%) ofcompound 47 as a white solid.

[0190] Synthesis of Compound 48

[0191] Lithium aluminum hydride (42 mg, 1.04 mmol) was added to an icecooled solution of the azide 47 (415 mg, 1.04 mmol) in 5.2 ml of Et₂Ounder argon. The reaction was allowed to warm to room temperature. After2 hours the solution was cooled in ice, diluted with 25 ml of diethylether and slowly quenched with 2 ml of saturated Na₂SO₄ solution. After10 minutes a white precipitate had formed and the solution was dilutedwith 50 ml of ethyl acetate, washed with 3×10 ml of brine, dried overMgSO₄, filtered and concentrated. The crude material was purified usinga column of silica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washingwith 5% MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, elutedwith 5% MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give a whitefoam which was shown by ¹H nmr to contain a trace of Et₃N. The materialwas taken up in 50 ml of hexanes, washed with 2×20 ml of brine, driedover MgSO₄, filtered and concentrated to give 322 mg (83%) of compound48 as a white foam.

[0192] Synthesis of Compound 49

[0193] A solution of the 3α-amino compound 48 (317 mg, 0.850 mmol), 4 MHCl in dioxane (255 μl, 1.02 mmol), THF (13.6 ml) and water (3.4 ml) wasstirred at room temperature overnight. The solution was concentrated todryness, triturated with 3×10 ml portions of acetone, evaporating offthe acetone after each trituration. Concentration gave 301 mg (96%) ofcompound 49 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 334.16; C₂₁H₃₆NO₂.

Example 2 3-Amino-6,7-dihydroxy-17-methylidene Steroid

[0194] A further example of alkenes related to compound 49 is shown inScheme 2. Olefination of the ketone 45 using methyltriphenylphosphoniumbromide and KO^(t)Bu in THF gave the 17-methylidene compound 50.Azidation using ZnN₆.2py, PPh₃ and DIAD in toluene gave the 3α-azidocompound 51. Lithium aluminum hydride reduction in THF gave the 3α-aminocompound 52. Treatment with 80% acetic acid removed the acetonideprotecting group and formed the ammonium acetate salt 53. Alternativelycompound 52 was treated with hydrochloric acid in acetonitrile and waterto give the hydrochloride salt 54.

[0195] Synthesis of Compound 50

[0196] A solution of KO^(t)Bu (2.0 g, 16.9 mmol), MePPh₃Br (6.0 g, 16.8mmol) and 27 ml of THF was stirred at room temperature under argon.After 1 hour the ketone 45 (2.00 g, 5.52 mmol) was added to the yellowsolution and the resulting solution was heated at reflux for 1 hour. Thereaction was quenched with 50 ml of brine, diluted with 100 ml of EtOAc,separated and washed with 25 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 1:1EtOAc/hexanes afforded 1.90 g (95%) of compound 50 as a white solid.

[0197] Synthesis of Compound 51

[0198] DIAD (0.85 ml, 4.10 mmol) was added dropwise over 15 minutes to aroom temperature solution of the 30-hydroxyl compound 50 (739 mg, 2.05mmol), ZnN₆.2py (473 mg, 1.54 mmol), Ph₃P (1.075 g, 4.10 mmol) andtoluene (20 ml) under argon. After 4 hours the reaction mixture wasloaded onto a column of silica gel packed in 10% ethyl acetate/hexanesand eluted with 20% ethyl acetate/hexanes to afford 743 mg (94%) ofcompound 51 as a white foam.

[0199] Synthesis of Compound 52

[0200] Lithium aluminum hydride (77 mg, 1.93 mmol) was added to an icecooled solution of the 3α-azide 51 in 5 ml of THF and 5 ml of diethylether under argon. The reaction was allowed to warm to room temperature.After 4 hours the solution was cooled in ice, diluted with 25 ml ofdiethyl ether and slowly quenched with 5 ml of saturated Na₂SO₄solution. After 10 minutes a white precipitate had formed and thesolution was diluted with 50 ml of ethyl acetate, washed with 3×10 ml ofbrine, dried over MgSO₄, filtered and concentrated. The crude materialwas purified using a column of silica gel prepared by packing in 1%Et₃N/CH₂Cl₂ and washing with 5% MeOH/CH₂Cl₂. The crude material wasloaded in CH₂Cl₂, eluted with 5% MeOH/CH₂Cl₂ and then 95:5:2CH₂Cl₂:MeOH:Et₃N to give 636 mg (92%) of compound 52 as &white solid.

[0201] Synthesis of Compound 53

[0202] A solution of the 3α-amine 52 (287 mg, 0.799 mmol) and 10 ml of80% acetic acid was heated at 40° C. for 1 hour. The reaction mixturewas concentrated to give a white foam. Acetone (10 ml) was added and thesolution was sonicated to dissolve the material and then evaporated.Another 10 ml portion of acetone was added, sonicated and evaporated togive 301 mg (99%) of compound 53 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.19;C₂₀H₃₄NO₂.

[0203] Synthesis of Compound 54

[0204] A solution of 4 M HCl in dioxane was added to a solution of theamine 52 in 1 ml of acetonitrile and 50 μl of water. The resulting gummysolid was diluted with 2 ml of acetonitrile and stirred vigorously untila solid formed. The solid was filtered and dried to give 50 mg (63%) ofcompound 54. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 320.19; C₂₀H₃₄NO₂.

Example 3 3-Amino-6,7-dihydroxy-17-methylidine Steroid AlternativeSynthesis

[0205] Intermediate 52 was also synthesized by the alternate route shownin Scheme 3. Azidation of the alcohol 45 using ZnN₆.2py, PPh₃ and DIADin toluene gave the 3α-azido compound 55. Hydrogenation of the azide,using Pd on carbon as catalyst gave the amine 56. Olefination ofcompound 56 using methyltriphenylphosphonium bromide and KO^(t)Bu in THFgave the 17-methylidene compound 52.

[0206] Synthesis of Compound 55

[0207] DIAD (2.4 ml, 11.6 mmol) was added dropwise over 20 minutes to aroom temperature solution of the 3β-hydroxyl compound 45 (2.108 g, 5.81mmol), ZnN₆.2py (1.34 g, 4.36 mmol), Ph₃P (3.05 g, 11.6 mmol) andtoluene (58 ml) under argon. After being allowed to react overnight, thereaction mixture was loaded onto a column of silica gel and eluted with20% ethyl acetate/hexanes to afford 1.14 g (50%) of compound 55 as awhite foam.

[0208] Synthesis of Compound 56

[0209] A solution of the azide 55 (1.10 g, 2.84 mmol), 10% Pd on carbon(60 mg, 0.057 mmol) and 28 ml of ethyl acetate was stirred at roomtemperature overnight under hydrogen. The solution was filtered throughcelite eluting with ethyl acetate. Purification by radial chromatographyeluting with 95:5:2 CH₂Cl₂:MeOH:Et₃N gave 892 mg (81%) of compound 56 asa white solid.

[0210] Synthesis of Compound 52

[0211] A solution of KO^(t)Bu (175 mg, 1.48 mmol), MePPh₃Br (528 mg,1.48 mmol) and 3 ml of THF was stirred at room temperature under argon.After 1 hour the ketone 56 (100 mg, 0.277 mmol) was added to the yellowsolution and the resulting solution was allowed to stir at roomtemperature overnight. The reaction was quenched with 5 ml of water,diluted with 50 ml of EtOAc, separated and washed with 10 ml of brine,dried over MgSO₄, filtered and concentrated. Purification by radialchromatography eluting with 95:5:2 CH₂Cl₂:MeOH:Et₃N afforded 96 mg (97%)of compound 52 as a white solid.

Example 4 3-Amino-6,7-dihydroxy-17-fluroethylidene Steroid

[0212] Halogenated analogues related to compound 49 can be preparedusing halogenated olefination reagents. Scheme 4 outlines the synthesisof the 20-fluoro analogue 64. The hydroxyl in compound 45 was protectedby treatment with tert-butyldimethylsilyl chloride and imidazole indimethylformamide (DMF). Olefination of the ketone 57 using the anion oftriethyl 2-fluoro-2-phosphonoacetate gives a mixture of compound 58 andits geometric isomer. The compounds are separable using silica gelchromatography. Lithium aluminum hydride reduction of the ester in Et₂Ogave the allylic alcohol 59. Treatment with sulfur trioxide pyridinecomplex in THF followed by addition of lithium aluminum hydride affordsthe dehydroxylated compound 60. Tetrabutylammonium fluoride in THFremoves the protecting group from the 3-hydroxyl to give compound 61.Azidation using ZnN₆.2py, PPh₃ and DIAD in toluene gave the 3α-azidocompound 62. Lithium aluminum hydride reduction in THF gave the 3α-amine63. Treatment with HCl in THF and water deprotected the 6- and7-hydroxyls and formed the ammonium chloride salt 64.

[0213] Synthesis of Compound 57

[0214] A solution of the ketone 45 (4.73 g, 13.1 mmol), TBSCl (3.01 g,19.6 mmol), imidazole (2.67 g, 39.2 mmol) and DMF (52 ml) was stirred atroom temperature overnight. The white slurry was diluted with 250 ml ofEtOAc, washed with 2×50 ml of water and 50 ml of brine, dried overMgSO₄, filtered and concentrated to give 5.97 g (96%) of compound 57 aswhite solid.

[0215] Synthesis of Compound 58

[0216] Lithium bis(trimethylsilyl)amide (10.0 ml of a 1.0 M solution inTHF, 10.0 mmol) was added to a room temperature solution of(EtO)₂P(O)CHFCO₂Et (2.65 g, 10.5 mmol) in THF (22 ml) under argon. After1 hour a solution of the ketone 57. (2.50 g, 5.25 mmol) in THF (20 ml)was added and the resulting solution was heated at reflux for 4.5 hoursand then stirred at room temperature overnight. The reaction wasquenched with 1.5 ml of saturated NaHCO₃ solution and then partiallyconcentrated to remove most of the THF. The residue was diluted with 200ml of EtOAc, washed with 3×20 ml of brine, dried over MgSO₄, filteredand concentrated. The crude material was purified by columnchromatography, eluting with 2.5% then 5% EtOAc/hexanes to give 1.50 g(50%) of compound 58 as a white solid.

[0217] Synthesis of Compound 59

[0218] Lithium aluminum hydride (106 mg, 2.66 mmol) was added to an icecooled solution of the ester 58 (1.50 g, 2.66 mmol) in Et₂O (13 ml)under argon. The solution was allowed to warm to room temperature. After3 hours the solution was cooled in ice and 20 ml of saturated Na₂SO₄solution was slowly added. After 10 minutes the solution was dilutedwith 150 ml of EtOAc, washed with water and brine, dried over MgSO₄,filtered and concentrated to give 1.44 g (quantitative) of compound 59as a white foam.

[0219] Synthesis of Compound 60

[0220] Sulfur trioxide pyridine complex (69.5 mg, 0.428 mmol) was addedto an ice cooled solution of the allylic alcohol 59 (149 mg, 0.285 mmol)in THF (2.8 ml) under argon. After 6 hours lithium aluminum hydride (68mg, 1.71 mmol) was added and the solution was allowed to warm to roomtemperature overnight. The solution was cooled in ice and 5 ml ofsaturated Na₂SO₄ solution was slowly added. After 10 minutes thesolution was diluted with 75 ml of EtOAc, washed with water and brine,dried over MgSO₄, filtered and concentrated to give 109 mg (76%) ofcompound 60 as a white solid.

[0221] Synthesis of Compound 61

[0222] A solution of compound 60 (410 mg, 0.810 mmol), Bu₄NF (0.89 ml ofa 1.0 M solution in THF, 0.89 mmol) and THF (5 ml) was heated at refluxunder argon. After 1.5 hours the solution was cooled to roomtemperature, diluted with 75 ml of EtOAc, washed with 20 ml of water and2×20 ml of brine, dried over MgSO₄, filtered and concentrated. Theresidue was filtered through silica gel eluting with EtOAc andconcentrated to give 318 mg (100%) of compound 61 as a white solid.

[0223] Synthesis of Compound 62

[0224] DIAD (0.33 ml, 1.59 mmol) was added dropwise over 10 minutes to aroom temperature solution of the 3β-alcohol 61 (312 mg, 0.796 mmol),ZnN₆.2py (183 mg, 0.597 mmol), Ph₃P (417 mg, 1.59 mmol) and toluene (8.0ml) under argon. After 3 hours the reaction mixture was loaded onto acolumn of silica gel packed in 10% EtOAc/hexanes and eluted with 20%EtOAc/hexanes to afford 322 mg (97%) of compound 62 as a crystallinesolid.

[0225] Synthesis of Compound 63

[0226] Lithium aluminum hydride (29 mg, 0.75 mmol) was added to an icecooled solution of the azide 62 (314 mg, 0.753 mmol) in 7.5 ml of Et₂Ounder argon. The reaction was allowed to warm to room temperature whilestirring overnight. The solution was cooled in ice and slowly quenchedwith 10 ml of saturated Na₂SO₄ solution. After 10 minutes a whiteprecipitate had formed and the solution was diluted with 75 ml of EtOAc,washed with 20 ml of water and 2×20 ml of brine, dried over MgSO₄,filtered and concentrated. The crude material was purified using acolumn of silica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washingwith 5% MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, elutedwith 5% MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give a whitesolid. ¹H NMR analysis indicated the material contained a trace of Et₃Ntherefore the material was taken up in 75 ml of CH₂Cl₂ and washed with2×25 ml of water, dried over MgSO₄, filtered and concentrated to give137 mg (47%) of compound 63 as a colorless film.

[0227] Synthesis of Compound 64

[0228] A solution of the 3α-amino compound 63 (137 mg, 0.35 mmol), 4 MHCl in dioxane (105 μl, 0.42 mmol), THF (5.6 ml) and water (1.4 ml) wasstirred at room temperature overnight. The solution was concentrated,the residue was twice taken up in 3 ml of methanol and concentrated togive 130 mg (96%) of compound 64 as an off-white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 352.14;C₂₁H₃₅FNO₂.

Example 5 3-Amino-6,7-dihydroxy-17-carbomethoxyethylidene Steroid

[0229] Olefination of compounds related to compound 57 can also becarried out to generate 21-carboalkoxy substituted analogues. Scheme 5shows the synthesis of the 21-carbomethoxy substituted example compound69. Olefination of the ketone 57 using the anion of trimethyl2-phosphonoacetate gives a mixture of compound 65 and its geometricisomer. The compounds are separable using silica gel chromatography.Tetrabutylammonium fluoride in THF removes the protecting group from the3-hydroxyl to give compound 66. Azidation using ZnN₆.2py, PPh₃ and DIADin toluene gave the 3α-azido compound 67. Hydrogenation of the azide,using Pd on carbon as catalyst gave the 3α-amine 68. Treatment with 80%acetic acid deprotected the 6- and 7-hydroxyls and formed the ammoniumacetate salt 69.

[0230] Synthesis of Compound 65

[0231] Lithium bis(trimethylsilyl)amide (2.00 ml of a 1.0 M solution inTHF, 2.00 mmol) was added to a room temperature solution of(MeO)₂P(O)CH₂CO₂Me (390 mg, 2.10 mmol) in THF (22 ml) under argon. After3 hours a solution of the ketone 57 (509 mg, 1.07 mmol) in THF (2 ml)was added and the resulting solution was heated at reflux for 3 days.The reaction was quenched with 5 ml of water, diluted with 75 ml ofEtOAc, washed with 2×15 ml of brine, dried over MgSO₄, filtered andconcentrated. The crude material was purified by column chromatography,eluting with 5% EtOAc/hexanes to give 307 mg (54%/o) of compound 65 as acolorless film. Also isolated was 119 mg (21%) of the Z-isomer.

[0232] Synthesis of Compound 66

[0233] A solution of 65 (296 mg, 0.550 mmol), Bu₄NF (0.61 ml of a 1.0 Msolution in THF, 0.61 mmol) and THF (3 ml) was heated at reflux underargon. After 1 hour the solution was cooled to room temperature, dilutedwith 20 ml of EtOAc, washed with 10 ml of water and 2×10 ml of brine,dried over MgSO₄, filtered and concentrated to give 230 mg (100%) ofcompound 66 as a white solid.

[0234] Synthesis of Compound 67

[0235] DIAD (252 μl, 1.28 mmol) was added dropwise to a room temperaturesolution of the 3β-alcohol 66 (230 mg, 0.55 mmol), ZnN₆.2py (147 mg,0.48 mmol), Ph₃P (335 mg, 1.28 mmol) and toluene (6.4 ml) under argon.After 2 hours the reaction mixture was purified by radialchromatography, eluting with 15% EtOAc/hexanes to afford 203 mg (84%) ofcompound 67.

[0236] Synthesis of Compound 68

[0237] A solution of the azide 67 (203 mg, 0.45 mmol), 10% Pd on carbon(48 mg, 0.045 mmol) and 4.5 ml of EtOAc was stirred at room temperatureunder hydrogen for 3 days. The solution was filtered through celiteeluting with EtOAc and MeOH to give 165 mg (88%) of compound 68.

[0238] Synthesis of Compound 69

[0239] A solution of the amine 68 (165 mg, 0.40 mmol) and 2 ml of 80%acetic acid was heated at 40° C. for 1 hour. The reaction mixture wasdiluted with 10 ml of toluene then concentrated to remove residualacetic acid. Trituration of the residue in 10 ml of cyclohexane,followed by filtration and drying gave 117 mg (67%) of compound 69 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 378.17; C₂₂H₃₆NO₄.

Example 6 3α-Amino-6α,7β-dihydroxyandrostan-17-one Acetic Acid Salt

[0240] Analogous methodology can be used to obtain compounds withdifferent functionalities at C17. For example a 17-ketone substitutedcompound is obtained by treatment of compound 56 with 80% acetic acid togive 3α-amino-6α,7β-dihydroxyandrostan-17-one acetic acid salt (70) (seeTable 2).

[0241] Synthesis of Compound 70

[0242] A solution of the ketone 56 (67 mg, 0.16 mmol) and 1 ml of 80%acetic acid was heated at 40° C. for 1 hour. The reaction mixture wasdiluted with 10 ml of toluene then concentrated to give 63 mg (100%) ofcompound 70. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 322.18; C₁₉H₃₂NO₃.

Example 7 3-Amino-6,7-dihydroxy-17-hydroxy Steroid

[0243] 17-Hydroxyl substituted analogues can be prepared from ketonesrelated to compound 56 as shown in Scheme 6. The carbonyl in compound 56was reduced with NaBH₄ in methanol to give exclusively the 17β-hydroxylisomer 71. Treatment with 80% acetic acid removed the acetonideprotecting group and formed the ammonium acetate salt 72.

[0244] Synthesis of Compound 71

[0245] An ice cooled solution of the ketone 56 (100 mg, 0.28 mmol),NaBH₄ (16 mg, 0.41 mmol) and 1.4 ml of MeOH was allowed to react for 2.5hours. The reaction was quenched by the addition of 1 ml of water andconcentrated to remove most of the MeOH. The residue was diluted with 40ml of CH₂Cl₂ and washed with 2×10 ml of brine, dried over MgSO₄,filtered and concentrated to give 90 mg (90%) of compound 71.

[0246] Synthesis of Compound 72

[0247] A solution of the amine 71 (90 mg, 0.25 mmol) and 2 ml of 80%acetic acid was heated at 40° C. for 2 hours. The reaction mixture wastwice diluted with 10 ml of toluene and concentrated to remove residualacetic acid. The residue was dissolved in 1 ml of MeOH and 5 ml ofhexanes, concentrated and dried to give 86 mg (90%) of compound 72 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 324.19; C₁₉H₃₄NO₃.

Example 8 Salts of 3α-Amino-6,7-dihydroxy-17-methylidene Steroid

[0248] The 6- and 7-hydroxyls can be protected using a variety ofprotecting groups. Suitable protecting groups are listed in Greene andWuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, NewYork, N.Y. (1999). Scheme 7 shows examples of analogues that have beensynthesized with the 6- and 7-hydroxyls protected as methyl ethers. Thestarting material compound 73 for the synthesis is described in U.S.Pat. No. 6,046,185. Generation of the dianion of compound 73 using NaHin dimethylformamide followed by alkylation with methyl iodide gavecompound 74. Treatment with 80% acetic acid removed both the cyclicketal and tert-butyldimethylsilyl ether protecting groups. Olefinationof compound 75 using methyltriphenylphosphonium bromide and KO^(t)Bu inTHF gave the 17-methylidene compound 76. Azidation using ZnN₆.2py, PPh₃and DIAD in toluene gave the 3α-azido compound 77. Lithium aluminumhydride reduction in THF gave the 3α-amine 78. Treatment with HCl inEt₂O and MeOH formed the ammonium chloride salt 79. Treatment ofcompound 78 with acetic acid formed the ammonium acetate salt 80.

[0249] Synthesis of Compound 74

[0250] Sodium hydride (0.50 g, 12.4 mmol) was added to a roomtemperature solution of the diol 73 (1.49 g, 3.10 mmol) in 15 ml of DMFunder nitrogen. After 2 hours the solution was cooled in ice and MeI(1.93 ml, 30.9 mmol) was added dropwise over 30 seconds. The reactionwas allowed to warm to room temperature while stirring overnight. Thereaction mixture was diluted with 100 ml of Et₂O, washed with 10 ml ofwater and 2×10 ml of brine, dried over MgSO₄, filtered and concentratedto give 1.73 g of crude compound 74 as a pale yellow oil.

[0251] Synthesis of Compound 75

[0252] A solution of crude compound 74 (1.73 g, 3.10 mmol) and 15 ml of80% acetic acid was stirred at room temperature for 4 hours. Thesolution was concentrated, the residue taken up in 50 ml of EtOAc,washed with 2×20 ml of saturated NaHCO₃ solution and 2×10 ml of brine,dried over MgSO₄, filtered and concentrated to give 1.18 g of crudecompound 75 as a white foam.

[0253] Synthesis of Compound 76

[0254] A solution of KO^(t)Bu (1.09 g, 9.20 mmol), MePPh₃Br (3.30 g,9.20 mmol) and 15 ml of THF was stirred at room temperature undernitrogen. After 2 hours crude ketone 75 (1.17, mg, 3.08 mmol) was addedto the yellow solution and the resulting solution was allowed to stir atroom temperature overnight. The reaction was quenched with 2 ml ofwater, diluted with 100 ml of EtOAc, washed with 3×10 ml of brine, driedover MgSO₄, filtered and concentrated. Column chromatography elutingwith 80% EtOAc/hexanes afforded 890 mg of impure compound 76 as a whitesolid.

[0255] Synthesis of Compound 77

[0256] DIAD (1.05 ml, 5.08 mmol) was added dropwise over 10 minutes to aroom temperature solution of the 3β-alcohol 76 (885 mg, 2.54 mmol),ZnN₆.2py (585 mg, 1.90 mmol), Ph₃P (1.33 g, 5.08 mmol) and toluene (25ml) under argon. After 11 hours the reaction mixture was purified bycolumn chromatography eluting with 15% EtOAc/hexanes to afford 594 mg(63%) of compound 77 as a crystalline solid.

[0257] Synthesis of Compound 78

[0258] Lithium aluminum hydride (0.79 ml of a 1 M solution in Et₂O, 0.79mmol) was added to an ice cooled solution the azide 77 (588 mg, 1.57mmol) in 15.7 ml of Et₂O under argon. After 10 minutes the reaction wasallowed to warn to room temperature while stirring overnight. After 1hour the reaction mixture was cooled in ice and slowly quenched with 10ml of saturated Na₂SO₄ solution. After 10 minutes a white precipitatehad formed and the liquid was decanted off. The residue was washed with2×25 ml of EtOAc and the washings were combined with the previouslydecanted ether solution. The solution was washed with 3×10 ml of brine,dried over MgSO₄, filtered and concentrated. Purification by columnchromatography eluting with 95:5:2 CH₂Cl₂:MeOH:Et₃N gave 434 mg (79%) ofcompound 78 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂.

[0259] Synthesis of Compound 79

[0260] Hydrogen chloride (0.26 ml of a 1.0 M solution in Et₂O, 0.26mmol) was added to a solution of the amine 78 (60 mg, 0.17 mmol) in 2 mlof Et₂O. The resulting gel-like material was dissolved in 5 ml ofmethanol and concentrated. The residue was dissolved in 1 ml ofmethanol, diluted with 5 ml of cyclohexane and concentrated to give 66mg (100%) of compound 79 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂.

[0261] Synthesis of Compound 80

[0262] A solution of the amine 78 (61 mg, 0.17 mmol) and 1 ml of aceticacid was allowed to stand at room temperature for 30 minutes. Thesolution was diluted with 5 ml of toluene and concentrated. The residuewas taken up in 5 ml of hexanes, concentrated and the residue was driedfor 2 hours using an Abderhalden drying apparatus with refluxing acetoneto give 71 mg (100%) of compound 80 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 348.20;C₂₂H₃₈NO₂.

Example 9 Salts of 3β-Amino-6,7-dihydroxy-17-methylidene Steroid

[0263] The stereochemistry at C3 can be inverted to give 3β-ammoniumsalt derivatives of any number of compounds related to compound 49. Thestereochemistry at C3 can be inverted in 3 synthetic steps as shown inScheme 8 for the synthesis of compounds 28 and 83. The 3β-hydroxylcompound 46 is converted to the 3β-mesylate 81 using methanesulfonylchloride and pyridine. Heating compound 81 and cesium acetate in 100° C.DMF gives the 3α-acetate compound 82. The inversion sequence iscompleted by methanolysis of the acetate in compound 82 using sodiummethoxide to give the 3α-hydroxyl compound 25. Treatment of compound 25with ZnN₆.2py, triphenylphosphine and DIAD in toluene produced the3β-azido compound 26. Lithium aluminum hydride reduction of the azide inEt₂O provided the 3β-amino compound 27. Treatment with HCl in THF andwater removes the acetonide group and forms the ammonium chloride salt28. Similarly, treatment of compound 27 with 80% acetic acid removes theacetonide group and forms the ammonium acetate salt 83. Using themethods outlined in Scheme 8, compound 50 is converted into compound 89and compound 61 is converted into compound 95 (see Table 1). Compounds26, 27, 87, 88, 89, 93, 94 and 95 are examples of compounds of theinvention having 3β stereochemistry.

[0264] Synthesis of Compound 81

[0265] Methanesulfonyl chloride (1.2 ml, 16 mmol) was added to an icecooled solution of the 3β-hydroxyl compound 46 (3.0 g, 8.0 mmol) inpyridine (20 ml) under argon. After 4 hours the solution was cooled inice and 20 ml of saturated NaHCO₃ solution was added. After 15 minutesthe solution was diluted with 150 ml of EtOAc and washed with 3×25 ml ofbrine, dried over MgSO₄, filtered and concentrated to give 3.6 g (100%)of compound 81 as an off-white foam.

[0266] Synthesis of Compound 82

[0267] A solution of the mesylate 81 (3.6 g, 8.0 mmol), cesium acetate(4.6 g, 24 mmol) and 40 ml of DMF was heated at 100° C. for 24 hours.The solution was diluted with 100 ml of water, extracted with 2×100 mlof Et₂O, washed with 2×50 ml of brine, dried over MgSO₄, filtered andconcentrated to give approximately 3 g of crude compound 82.

[0268] Synthesis of Compound 25

[0269] A solution of Na (398 mg, 17.3 mmol) in MeOH (21.5 ml) was addedto the 3α-acetate 82 (1.8 g, 4.3 mmol) in THF (10 ml). After 2 hours, 20ml of water was added and the resulting solution was diluted with 100 mlof EtOAc, washed consecutively with saturated NaHCO₃ solution, water andbrine, dried over MgSO₄, filtered and concentrated to give 1.58 g (98%)of crude compound 25 as a yellow foam.

[0270] Synthesis of Compound 26

[0271] DIAD (1.70 ml, 8.24 mmol) was added dropwise over 10 minutes to aroom temperature solution of the 3α-alcohol 25 (1.54 g, 4.12 mmol),ZnN₆.2py (0.94 g, 3.09 mmol), Ph₃P (2.16 g, 8.24 mmol) and toluene (44ml) under argon. After stirring overnight, the reaction mixture wasloaded onto a column of silica gel packed in 10% EtOAc/hexanes andeluted with 10% EtOAc/hexanes to afford 0.89 g (61%) of compound 26 as awhite solid.

[0272] Synthesis of Compound 27

[0273] Lithium aluminum hydride (146 mg, 3.66 mmol) was added to an icecooled solution the azide 26 (1.46 g, 3.66 mmol) in 18.3 ml of Et₂Ounder argon. The reaction was allowed to warm to room temperature. After1.5 hours the solution was cooled in ice, diluted with 25 ml of Et₂O andslowly quenched with 20 ml of saturated Na₂SO₄ solution. After 10minutes a white precipitate had formed and the solution was diluted with50 ml of EtOAc, washed with 3×10 ml of brine, dried over MgSO₄, filteredand concentrated to give 1.31 g (96%) of compound 27 as a white foam.

[0274] Synthesis of Compound 28

[0275] A solution of the 3β-amino compound 27 (227 mg, 0.609 mmol), 4 MHCl in dioxane (183 μl, 0.73 mmol), THF (9.7 ml) and water (2.4 ml) wasstirred at room temperature overnight. Evaporated the THF and water,took up the residue in 5 ml of methanol and concentrated, trituratedwith 5 ml of acetone, concentrated to give 224 mg (100%) of compound 28as a white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAcin 4:1 water and MeCN) 334.10; C₂₁H₃₆NO₂.

[0276] Synthesis of Compound 83

[0277] A solution of the amine 27 (412 mg, 1.10 mmol) and 5 ml of 80%acetic acid was stirred at room temperature for 4 hours. The reactionmixture was diluted with 5 ml of toluene then concentrated. The residuewas twice more taken up in 5 ml portions of toluene and concentrated toremove residual acetic acid. The residue was twice triturated in 10 mlof CH₂Cl₂ and concentrated to give 430 mg (99%) of compound 83 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 334.19; C₂₁H₃₆NO₂.

[0278] Synthesis of Compound 84

[0279] Methanesulfonyl chloride (0.33 ml, 4.2 mmol) was added to an icecooled solution of the 3β-hydroxyl compound 50 (754 mg, 2.09 mmol) inpyridine (5.3 ml) under argon. After 4 hours the solution was cooled inice and 5 ml of saturated NaHCO₃ solution was added. After 15 minutesthe solution was diluted with 60 ml of ethyl acetate and washed with 3times with brine, dried over MgSO₄, filtered and concentrated to give860 mg (94%) of compound 84 as an off-white solid

[0280] Synthesis of Compound 85

[0281] A solution of the mesylate 84 (860 mg, 1.96 mmol), cesium acetate(1.13 g, 5.88 mmol) and 10 ml of DMF was heated at 95° C. for 32 hours.The solution was diluted with 50 ml of water, extracted with 2×100 ml ofEt₂O, washed with 2×30 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 5% and8% EtOAc/Hexanes afforded 558 mg (71%) of compound 85 as white solid.

[0282] Synthesis of Compound 86

[0283] A solution of Na (128 mg, 5.56 mmol) in MeOH (7 ml) was added tothe 3α-acetate 85 (558 mg, 1.38 mmol). After 2 hours 5 ml of saturatedNaHCO₃ solution was added and the resulting solution was diluted with100 ml of EtOAc. The solution washed with 2×20 ml of water and 2×20 mlof brine, dried over MgSO₄, filtered and concentrated to give 491 mg(99%) of compound 86 as a white solid.

[0284] Synthesis of Compound 87

[0285] DIAD (0.57 ml, 2.74 mmol) was added dropwise over 15 minutes to aroom temperature solution of the 3α-hydroxy compound 86 (493 mg, 1.37mmol), ZnN₆.2py (315 mg, 1.03 mmol), Ph₃P (718 mg, 2.74 mmol) andtoluene (13.7 ml) under argon. After 3.5 hours the reaction mixture wasloaded onto a column of silica gel packed in 10% ethyl acetate/hexanesand eluted with 10% ethyl acetate/hexanes to afford 390 mg (74%) ofcompound 87 as a viscous oil.

[0286] Synthesis of Compound 88

[0287] Lithium aluminum hydride (40 mg, 1.01 mmol) was added to an icecooled solution of the azido compound 87 (390 mg, 1.01 mmol) in 5 ml ofdiethyl ether under argon. The reaction was allowed to warm to roomtemperature. After 2 hours the solution was cooled in ice, diluted with25 ml of diethyl ether and slowly quenched with 2 ml of saturated Na₂SO₄solution. After 10 minutes a white precipitate had formed and thesolution was diluted with 40 ml of ethyl acetate, washed with 3×15 ml ofbrine, dried over MgSO₄, filtered and concentrated. The crude materialwas purified using a column of silica gel prepared by packing in 1%Et₃N/CH₂Cl₂ and washing with 5% MeOH/CH₂Cl₂. The crude material wasloaded in CH₂Cl₂, eluted with 5% MeOH/CH₂Cl₂ and then 95:5:2CH₂Cl₂:MeOH:Et₃N to give 277 mg (76%) of compound 88 as a white solid.

[0288] Synthesis of Compound 89

[0289] A solution of the amino compound 88 (270 mg, 0.752 mmol) and 10ml of 80% acetic acid was heated at 40° C. for 1 hour. The reactionmixture was concentrated to give a white foam. Acetone (10 ml) was addedand sonicated to dissolve the material and then evaporated. Another 10ml portion of acetone was added, sonicated and evaporated to give 285 mg(100%) of compound 89 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.26; C₂₀H₃₄NO₂.

[0290] Synthesis of Compound 90

[0291] Methanesulfonyl chloride (0.20 ml, 2.56 mmol) was added to an icecooled solution of the 3β-hydroxyl compound 61 (501 mg, 1.28 mmol) inpyridine (3.2 ml) under argon. After 4 hours the solution was cooled inice and 5 ml of saturated NaHCO₃ solution was added. After 15 minutesthe solution was diluted with 50 ml of EtOAc and washed 3 times withbrine, dried over MgSO₄, filtered and concentrated to give 590 mg (98%)of compound 90 as a white foam.

[0292] Synthesis of Compound 91

[0293] A solution of the mesylate 90 (590 mg, 1.25 mmol), cesium acetate(722 mg, 3.76 mmol) and 6.2 ml of DMF was heated at 100° C. for 24hours. The solution was diluted with 50 ml of water, extracted with 2×50ml of Et₂O, washed with 2×30 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 8%EtOAc/hexanes afforded 297 mg (55%) of compound 91 as a white solid.

[0294] Synthesis of Compound 92

[0295] A solution of Na (63 mg, 2.7 mmol) in MeOH (3.4 ml) was added tothe 3α-acetate 91 (297 mg, 0.684 mmol) in THF (1 ml). The solution wasstirred overnight, 5 ml of water and 80 ml of EtOAc were added andwashed twice with water and twice with brine, dried over MgSO₄, filteredand concentrated to give 251 mg (94%/o) of compound 92 as a white solid.

[0296] Synthesis of Compound 93

[0297] DIAD (0.26 ml, 1.24 mmol) was added dropwise over 10 minutes to aroom temperature solution of the 3α-alcohol 92 (243 mg, 0.620 mmol),ZnN₆.2py (143 mg, 0.465 mmol), Ph₃P (325 mg, 1.24 mmol) and toluene (6.2ml) under argon. After 4 hours the reaction mixture was loaded onto acolumn of silica gel packed in 10% EtOAc/hexanes and eluted with 20%EtOAc/hexanes to afford 209 mg of impure compound 93 as a yellow oil.

[0298] Synthesis of Compound 94

[0299] Lithium aluminum hydride (20 mg, 0.50 mmol) was added to an icecooled solution the impure azide 93 (209 mg, 0.50 mmol) in 5 ml of Et₂Ounder argon. The reaction was allowed to warm to room temperature. After4 hours the solution was cooled in ice, diluted with 25 ml of Et₂O andslowly quenched with 2 ml of saturated Na₂SO₄ solution. After 10 minutesa white precipitate had formed and the solution was diluted with 50 mlof EtOAc, washed with 3×10 ml of brine, dried over MgSO₄, filtered andconcentrated. The crude material was chromatographed using a column ofsilica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washing with 5%MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, eluted with 5%MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give 97 mg of impurecompound 94 as a white solid.

[0300] Synthesis of Compound 95

[0301] A solution of the impure 3β-amino compound 94 (97 mg, 0.25 mmol),4 M HCl in dioxane (74 μl, 0.30 mmol), THF (4 ml) and water (1 ml) wasstirred at room temperature. After 4 hours the solution wasconcentrated, the residue was taken up in 5 ml of methanol andconcentrated. The residue was twice triturated with 5 ml of acetone andconcentrated. The white solid was dissolved in approximately 0.5 ml ofwater and acetone (5 ml) was slowly added until crystals appeared. Thecrystals were filtered, rinsed with acetone and dried to give 66 mg ofcompound 95 as colorless fine needles. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 352.09;C₂₁H₃₅FNO₂.

Example 10 3-Amino-6,7-Dihydroxy-17-Alkyl Steroid

[0302] Any compounds having the 17(20)-alkenyl functionality can havethe double bond hydrogenated using H₂ in the presence of a catalyst suchas 10% Pd on carbon. For example compound 96 has been prepared fromcompound 28 as shown in Scheme 9. Similarly, compound 97 was preparedfrom compound 49 using the same methodology as shown in Scheme 9 (seeTable 2).

[0303] Synthesis of Compound 96

[0304] A solution of the olefin 28 (52 mg, 0.14 mmol), 10% Pd on carbon(15 mg, 0.014 mmol) and methanol (3 ml) was stirred at room temperatureovernight under hydrogen. The solution was filtered through celiteeluting with 50 ml of methanol and concentrated to give 50 mg (96%) ofcompound 96 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 336.24; C21H₃₈NO₂.

[0305] Synthesis of Compound 97

[0306] A solution of the olefin 49 (844 mg, 2.28 mmol), 10% Pd on carbon(243 mg, 0.228 mmol) and methanol (11 ml) was stirred at roomtemperature overnight under hydrogen. The solution was filtered throughcelite eluting with 50 ml of methanol and concentrated. The residue wastriturated in 10 ml of acetone, filtered and dried to give 801 mg (94%)of compound 97 as a white solid. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 336.21; C₂₁H₃₈NO₂.

Example 11 3-Secondary Amino-6,7-Dihydroxy-17-Methylidene Steroid

[0307] Any amine related to compound 52 can be coupled to an aldehyde orketone to prepare secondary or tertiary amines. Reaction of compound 52with a solution of 4-isopropylbenzaldehyde and titanium isopropoxide inTHF followed by reduction with sodium borohydride gives compound 99.Treatment with 80% acetic acid removes the acetonide group and forms theammonium acetate salt 100. Example compounds 101-107 were synthesizedusing the methods outlined in Scheme 10 (see Table 6).

[0308] Synthesis of Compound 99

[0309] Titanium(IV) isopropoxide (120 μl, 0.42 mmol) was added to a roomtemperature solution of the amine 52 (100 mg, 0.28 mmol),4-isopropylbenzaldehyde (46 μl, 0.31 mmol) and 1.4 ml of THF undernitrogen. After 12 hours a solution of NaBH₄ (29 mg, 0.78 mmol) in 1 mlof EtOH was added and the reaction was continued for another 8 hours.The reaction was quenched by the addition of 3 ml of brine, diluted with30 ml of EtOAc, separated, washed with 10 ml of brine, dried over MgSO₄,filtered and concentrated. Purification using radial chromatographyafforded 50 mg (36%) of compound 99.

[0310] Synthesis of Compound 100

[0311] A solution of the amine 99 (50 mg, 0.10 mmol) and 1 ml of 80%acetic acid was heated at 40° C. for 3 hours. The reaction mixture wastwice taken up in 5 ml portions of toluene and concentrated and thenonce each with acetone and hexanes to give 25 mg (51%) of compound 100.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 452.27; C₃₀H₄₆NO₂.

[0312] Synthesis of Compound 101

[0313] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with 2-fluorobenzaldehyde(32 μl, 0.32 mmol) to give 43 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 49 mg (37%) of compound 101 asa white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 428.22; C₂₇H₃₉FNO₂.

[0314] Synthesis of Compound 102

[0315] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with3-(trifluoromethyl)benzaldehyde (41 μl, 0.31 mmol) to give 61 mg ofamine intermediate. The amine intermediate was treated with 1 ml of 80%acetic acid at 40° C. for 3 hours. The reaction mixture was diluted with5 ml of toluene and concentrated. The residue was dissolved in 1 ml ofacetone, diluted with 5 ml of hexanes and concentrated to give 64 mg(45%/o) of compound 102 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 478.18;C₂₈H₃₉F₃NO₂.

[0316] Synthesis of Compound 103

[0317] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with o-anisaldehyde (42 mg,0.31 mmol) to give 30 mg of amine intermediate. The amine intermediatewas treated with 1 ml of 80% acetic acid at 40° C. for 3 hours. Thereaction mixture was diluted with 5 ml of toluene and concentrated. Theresidue was dissolved in 1 ml of acetone, diluted with 5 ml of hexanesand concentrated to give 18 mg (14%) of compound 103. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 440.23;C₂₈H₄₂NO₃.

[0318] Synthesis of Compound 104

[0319] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with4-(trifluoromethoxy)benzaldehyde (44 μl, 0.31 mmol) to give 86 mg ofamine intermediate. The amine intermediate was treated with 1.5 ml of80% acetic acid at 40° C. for 3 hours. The reaction mixture was dilutedwith 5 ml of toluene and concentrated. The residue was dissolved in 1 mlof acetone, diluted with 5 ml of hexanes and concentrated to give 84 mg(57%) of compound 104 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 494.15;C₂₈H₃₉F₃NO₃.

[0320] Synthesis of Compound 105

[0321] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with 3-phenoxybenzaldehyde(60 mg, 0.32 mmol) to give 73 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 87 mg (58%) of compound 105 asa white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 502.20; C₃₃H₄₄NO₃.

[0322] Synthesis of Compound 106

[0323] Using the procedure described for the synthesis of compound 99,the amine 52 (100 mg, 0.28 mmol) was reacted with 3-nitrobenzaldehyde(46 mg, 0.31 mmol) to give 18 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 18 mg (14%) of compound 106 asan off-white solid. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 455.20; C₂₇H₃₉N₂O₄.

[0324] Synthesis of Compound 107

[0325] Using the procedure described for the synthesis of compound 99,the amine 52 (200 mg, 0.55 mmol) was reacted with3-pyridylcarboxaldehyde (82 μl, 0.61 mmol) to give 100 mg of amineintermediate. A suspension of the amine intermediate, 4 M HCl in dioxane(65 μl, 0.26 mmol), 110 μl of water and 2.2 ml of acetonitrile wasstirred at room temperature for 1 hour. The solution was filtered andthe solid was dried to afford 77 mg (30%) of compound 107 as a whitesolid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1water and MeCN) 411.21; C₂₆H₃₉N₂O₂.

Example 12 3-Cycloamino-6,7-dihydroxy-17-ethylidene Steroid

[0326] Any ketone related to compound 108 may be coupled to an amineusing the methodology shown in Scheme 11. The starting material compound108 for the synthesis is described in U.S. Pat. No. 6,046,185. Reactionof compound 108 with piperidine and sodium cyanoborohydride in methanolgave compound 109 as a mixture of isomers at C3. Treatment with 80%acetic acid removed the acetonide protecting group and formed theammonium acetate salt 110. Example compound 111 was synthesized usingthe methods outlined in Scheme 11, except hydrochloric acid is used inplace of acetic acid (see Table5). A 3-cycloamino group is a groupattached to the 3-position, where the carbon at the 3-position isattached directly to a nitrogen, and this nitrogen is part of aheterocyclic ring.

[0327] Synthesis of Compound 109

[0328] A solution of the ketone 108 (200 mg, 0.54 mmol), piperidine (266μl, 2.68 mmol), 100 mg of 3 Å molecular sieves, NaBH₃CN (24 mg, 0.38mmol) and 5.4 ml of MeOH was stirred at room temperature for 24 hours.The reaction mixture was diluted with 20 ml of water and extracted with2×20 ml of CH₂Cl₂. The combined extractions were washed with 10 ml ofbrine, dried over MgSO₄, filtered and concentrated. The crude materialwas purified using radial chromatography eluting with 20% MeOH/CH₂Cl₂ toafford 112 mg (47%) of compound 109 as a white solid.

[0329] Synthesis of Compound 110

[0330] A solution of the amines 109 (102 mg, 0.23 mmol) and 5 ml of 80%acetic acid was heated at 40° C. for 1 hour. The solution wasconcentrated, the residue was taken up in 2 ml of MeOH, diluted with 15ml of toluene and concentrated. The residue was triturated in 5 ml ofacetone, filtered and dried to give 44 mg (42%) of compound 110 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 402.31; C₂₆H₄₄NO₂.

[0331] Synthesis of Compound 111

[0332] Using the procedure described for the synthesis of compound 109,the ketone 108 (200 mg, 0.54 mmol) was reacted with morpholine (234 μl,2.68 mmol) to give 56 mg of the amine intermediate. The amineintermediate was treated with 5 ml of 80% acetic acid at 40° C. for 1hour. The solution was concentrated, dissolved in 5 ml of MeOH andconcentrated. ¹H and ¹³C NMR analyses indicated the acetonide protectinggroup had been removed but little or none of the salt had formed. Thematerial was treated with 4 M HCl in dioxane (32 μl, 0.13 mmol) and 2 mlof acetone giving a white precipitate. The suspension was diluted with 2ml of acetone, filtered and dried to give 48 mg (20%) of compound 111 asa white solid. LC/MS (direct inflision, electrospray +ve, 10 mM NH₄OAcin 4:1 water and MeCN) 404.20; C₂₅H₄₂NO₃.

Example 13 3-Oxo to 3-Secondary Amino Conversion in Steroid

[0333] Any ketone related to compound 108 can be coupled to an amineusing the methodology shown in Scheme 12. Methylamine is added to asolution of compound 108 and titanium isopropoxide in THF, followed byreduction with sodium borohydride. The solution is filtered and elutedthrough MP-TsOH resin to give compound 112, as a mixture of isomers atC3. Treatment with HCl in acetonitrile and water formed the ammoniumchloride salt 113. Example compounds 114-129 were synthesized using themethods outlined in Scheme 12, except that acetic acid was used in placeof hydrochloric acid for the examples in which ammonium acetate saltswere formed (see Table 5).

[0334] Synthesis of Compound 112

[0335] Titanium(IV) isopropoxide (270 μl, 0.92 mmol) was added to a roomtemperature solution of the ketone 108 (250 mg, 0.67 mmol), methylaminehydrochloride (41 mg, 0.61 mmol) and 1.5 ml of THF under nitrogen. After12 hours a solution of NaBH₄ (65 mg, 1.7 mmol) in 2.3 ml of EtOH wasadded and the reaction was continued for another 10 hours. The reactionwas quenched by the addition of 0.5 ml of water and filtered to remove awhite precipitate. The solution was loaded onto a column of 600 mg ofMP-TsOH resin and eluted with 3 ml of MeOH then 4 ml of 2 M NH₃ in MeOH.The NH₃/MeOH fraction was concentrated to give 76 mg (32%) of compound112.

[0336] Synthesis of Compound 113

[0337] A suspension of compound 112 (76 mg, 0.21 mmol), 4 M HCl indioxane (75 μl, 0.30 mmol), 50 μl of water and 1 ml of acetonitrile wasstirred at room temperature for 1 hour. The solution was filtered andthe solid was dried to afford 38 mg (13%) of compound 113 as a greysolid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1water and MeCN) 348.19; C₂₂H₃₈NO₂.

[0338] Synthesis of Compound 114

[0339] Using the procedures described for the synthesis of compound 112,the ketone 108 (200 mg, 0.53 mmol) was reacted with propylaminehydrochloride (47 mg, 0.49 mmol) to give 72 mg of amine intermediate.The amine intermediate was treated with 75 μl of the HCl solution togive 34 mg (17%) of compound 114. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 376.22; C₂₄H₄₂NO₂.

[0340] Synthesis of Compound 115

[0341] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with amylamine (70 μl,0.61 mmol) to give 82 mg of amine intermediate. The amine intermediatewas treated with 75 μl of the HCl solution to give 75 mg (28%) ofcompound 115 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 404.28; C₂₆H₄₆NO₂.

[0342] Synthesis of Compound 116

[0343] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with cyclopentylamine (60μl, 0.61 mmol) to give 99 mg of amine intermediate. The amineintermediate was treated with 200 μl of acetic acid for 1 hour and wastwice taken up and concentrated from 1 ml portions of toluene. Theresidue was triturated in 1 ml of cyclohexane, filtered and dried togive 98 mg (35%) of compound 116. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 402.27; C₂₆H₄₄NO₂.

[0344] Synthesis of Compound 117

[0345] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with cyclohexylamine (70μl, 0.61 mmol) to give 120 mg of amine intermediate. The amineintermediate was treated with 0.5 ml of acetic acid for 1 hour and wastwice taken up and concentrated from 1 ml portions of toluene. Theresidue was triturated in 1 ml of cyclohexane, filtered and dried togive 101 mg (32%) of compound l17. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 416.25; C₂₇H₄₆NO₂.

[0346] Synthesis of Compound 118

[0347] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with pyrrolidine (51 μl,0.61 mmol) to give 70 mg of amine intermediate. The amine intermediatewas treated with 50 μl of acetic acid for 1 hour and 1 ml of cyclohexanewas added to give a solid, which was filtered and dried to afford 65 mg(22%) of compound 118. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 388.28; C₂₅H₄₂NO₂.

[0348] Synthesis of Compound 119

[0349] Using the procedures described for the synthesis of compound 112,the ketone 108 (200 mg, 0.53 mmol) was reacted withN-propylethylenediamine (60 μl, 0.49 mmol) to give 99 mg of amineintermediate. The amine intermediate was treated with 75 μl of the HClsolution to give 47 mg (20%) of compound 119. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 419.32;C₂₆H₄₇N₂O₂.

[0350] Synthesis of Compound 120

[0351] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted withN,N-dimethylethylenediamine (65 μl, 0.61 mmol) to give 93 mg of amineintermediate. The amine intermediate was treated with 75 μl of the HClsolution to give 77 mg (29%) of compound 120 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)405.28; C₂₅H₄₅N₂O₂.

[0352] Synthesis of Compound 121

[0353] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with piperazine (52 mg,0.61 mmol) to give 33 mg of amine intermediate. The amine intermediatewas treated with 200 μl of acetic acid for 1 hour and was twice taken upand concentrated from 1 ml portions of toluene. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to give 39 mg(14%) of compound 121. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 403.23; C₂₅H₄₃N₂O₂.

[0354] Synthesis of Compound 122

[0355] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with ethanolamine (33 μl,0.61 mmol) to give 136 mg of amine intermediate. The amine intermediatewas treated with 75 μl of the HCl solution to give 124 mg (50%) ofcompound 122 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 378.19; C₂₃H₄₀NO₃.

[0356] Synthesis of Compound 123

[0357] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with 5-amino-1-pentanol(63 mg, 0.61 mmol) to give 129 mg of amine intermediate. The amineintermediate was treated with 75 μl of the HCl solution to give 65 mg(24%) of compound 123 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 420.25; C₂₆H₄₆NO₃.

[0358] Synthesis of Compound 124

[0359] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with2-(2-aminoethylamino)ethanol (62 μl, 0.61 mmol) to give 90 mg of amineintermediate. The amine intermediate was treated with 75 μl of the HClsolution to give 79 mg (28%) of compound 124 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)421.24; C₂₅H₄₅N₂O₃.

[0360] Synthesis of Compound 125

[0361] Using the procedures described for the synthesis of compound 112,the ketone 108 (200 mg, 0.53 mmol) was reacted with m-toluidine (52 μl,0.49 mmol) to give 95 mg of amine intermediate. The amine intermediatewas treated with 75 μl of the HCl solution to give 43 mg (19%) ofcompound 125. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 424.23; C₂₈H₄₂NO₂.

[0362] Synthesis of Compound 126

[0363] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with 4-aminophenol (67mg, 0.61 mmol) to give 138 mg of amine intermediate. The amineintermediate was treated with 75 μl of the HCl solution to give 41 mg(14%) of compound 126. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 426.18; C₂₇H₄₀NO₃.

[0364] Synthesis of Compound 127

[0365] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with sulfanilamide (105mg, 0.61 mmol) to give, after purification using radial chromatography,24 mg of amine intermediate. The amine intermediate was treated with 75μl of the HCl solution to give 23 mg (7%) of compound 127. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 489.17;C₂₇H₄₁N₂O₄S.

[0366] Synthesis of Compound 128

[0367] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with3-aminomethylpyridine (62 μl, 0.61 mmol) to give 108 mg of amineintermediate. The amine intermediate was reacted with 1 ml of 80% aceticacid at 40° C. for 1 hour. The reaction mixture was concentrated and wastwice taken up and concentrated from 1 ml portions of toluene. Theresidue was triturated in 1 ml of cyclohexane, filtered and dried togive 117 mg (41%) of compound 128. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 425.24; C₂₇H₄₁N₂O₂.

[0368] Synthesis of Compound 129

[0369] Using the procedures described for the synthesis of compound 112,the ketone 108 (250 mg, 0.67 mmol) was reacted with histamine (68 mg,0.61 mmol) to give 120 mg of amine intermediate. The amine intermediatewas reacted with 1 ml of 80% acetic acid at 40° C. for 1 hour. Thereaction mixture was concentrated and was twice taken up andconcentrated from 1 ml portions of toluene. The residue was trituratedin 1 ml of cyclohexane, filtered and dried to give 128 mg (38%) ofcompound 129. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 428.23; C₂₆H₄₂N₃O₂.

Example 14 3-Amino to 3-Acylamino Conversion in Steroid

[0370] Amide and sulfonamide analogues can be prepared from any aminerelated to compound 52. Scheme 13 shows the synthesis of the amide 131.Acetylation of the amine 52 in CH₂Cl₂, using acetyl chloride usingacetyl chloride and resin bound diethylamine gave the amide 130.Treatment with 80% acetic acid removed the acetonide group giving thedihydroxyamide 131.

[0371] Synthesis of Compound 130

[0372] A solution of the amine 52 (100 mg, 0.28 mmol), acetyl chloride(50 μl, 0.70 mmol), 440 mg of PS-DIEA resin and 2.4 ml of CH₂Cl₂ wasstirred at room temperature for 16 hours. The resin was filtered and thefiltrate was incubated for 2 hours with 260 mg of PS-Trisamine resin.The resin was filtered and the filtrate was concentrated. Purificationusing radial chromatography afforded 69 mg (62%) of compound 130.

[0373] Synthesis of Compound 131

[0374] A solution of the amide 130 (69 mg, 0.17 mmol) and 1 ml of 80%acetic acid was heated at 40° C. for 1 hour. The reaction mixture wastwice taken up and concentrated from 5 ml of toluene, once from 5 ml ofMeOH and once from 1 ml of acetone and 5 ml of hexanes to give 62 mg(62%) of compound 131 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 384.16;C₂₂H₃₅NNaO₃, 362.20; C₂₂H₃₆NO₃, 344.18; C₂₂H₃₄NO₂.

[0375] Synthesis of Compound 132

[0376] Using the procedure described for the synthesis of compound 130,the amine 52 (88 mg, 0.24 mmol) was reacted with benzoyl chloride (65μl, 0.56 mmol) to give 64 mg of amide intermediate. A solution of theamide intermediate and 2 ml of 80% acetic acid was heated at 40° C. for1 hour. The reaction mixture was twice taken up and concentrated from 5ml of toluene, once from 5 ml of MeOH and once from 1 ml of acetone and5 ml of hexanes to give 55 mg (55%) of compound 132 as a white, solid(see Table 3). LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 446.18; C₂₇H₃₇NNaO₃, 424.29; C₂₇H₃₈NO₃, 406.19;C₂₇H₃₆NO₂.

[0377] Synthesis of Compound 133

[0378] Using the procedure described for the synthesis of compound 130,the amine 52 (100 mg, 0.28 mmol) was reacted with isopropylsulfonylchloride (63 μl, 0.56 mmol) to give 38 mg of sulfonamide intermediate. Asolution of the sulfonamide intermediate and 1.5 ml of 80% acetic acidwas heated at 40° C. for 1 hour. The reaction mixture was twice taken upand concentrated from 5 ml of toluene, once from 5 ml of MeOH and oncefrom 1 ml of acetone and 5 ml of hexanes to give 35 mg (29%) of compound133 as an off-white solid (see Table 3). LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 426.14;C₂₃H₄₀NO₄S.

[0379] Synthesis of Compound 134

[0380] Using the procedure described for the synthesis of compound 130,the amine 52 (100 mg, 0.28 mmol) was reacted with benzenesulfonylchloride (90 μl, 0.70 mmol) to give 105 mg of sulfonamide intermediate.A solution of the sulfonamide intermediate and 2 ml of 80% acetic acidwas heated at 40° C. for 5 hours. The reaction mixture was twice takenup and concentrated from 5 ml of toluene, once from 5 ml of MeOH andonce from 1 ml of acetone and 5 ml of hexanes to give 83 mg (65%) ofcompound 134 as a white solid (see Table 3). LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 482.11;C₂₆H₃₇NNaO₄S, 477.17; C₂₆H₄₁N₂O₄S, 460.15; C₂₆H₃8NO₄S.

Example 15 3-Acylamiobiotin-6,7-hydroxy-17-ethylidene Steroid

[0381] Scheme 14 shows the synthesis of the amide 135. Reaction of theamine 83 with triethylamine and a water soluble version of biotin esterN-hydroxysuccinimide in methanol and water gave the biotinylated amideanalogue 135.

[0382] Synthesis of Compound 135

[0383] A solution of compound 83 (97 mg, 0.25 mmol), Et₃N (104 μl, 0.75mmol), sulfo-NHS-biotin (120 mg, 0.27 mmol), 2.5 ml of MeOH and 2.5 mlof water was stirred at room temperature overnight. The reaction mixturewas concentrated and purified using reverse phase column chromatographyeluting with 5% water/MeOH to afford 89 mg (64%) of compound 135 as anoff-white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAcin 4:1 water and MeCN) 560.30; C₃₁H₅₀N₃O₄S.

Example 16 3-Urea-6,7-hydroxy-17-methylidene Steroid

[0384] Any of the amines related to compound 52 can be reacted withisocyanates or isothiocyanates to give compounds having urea or thioureafunctionalities. Compounds 136, 137 and 138 are examples of ureas thatwere synthesized using the methods shown in Scheme 15 (see Table 3).

[0385] Synthesis of Compound 136

[0386] A solution of the amine 52 (100 mg, 0.28 mmol), phenyl isocyanate(76 μl, 0.70 mmol) and 2.4 ml of CH₂Cl₂ was stirred at room temperaturefor 16 hours. The solution was incubated for 2 hours with 260 mg ofPS-Trisamine resin. The resin was filtered and the filtrate wasconcentrated. Purification using radial chromatography gave 95 mg (71%)of compound 136.

[0387] Synthesis of Compound 137

[0388] A solution of the urea 136 (95 mg, 0.20 mmol) and 2 ml of 80%acetic acid was heated at 80° C. for 2 hours. The reaction mixture wastaken up and concentrated from 5 ml of toluene, from 5 ml of MeOH andfrom 5 ml of hexanes. Purification using radial chromatography elutingwith 95:5:2 CH₂Cl₂:MeOH:Et₃N afforded 40 mg (33%) of compound 137. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)461.18; C₂₇H₃₈N₂NaO₃, 439.22; C₂₇H₃₉N₂O₃, 421.25; C₂₇H₃₇N₂O₂.

[0389] Synthesis of Compound 138

[0390] Using the procedure described for the synthesis of compound 137,the amine 52 (100 mg, 0.28 mmol) was reacted with propyl isocyanate (52μl, 0.56 mmol) to give 72 mg of urea intermediate. A solution of theurea intermediate and 2 ml of 80% acetic acid was heated at 80° C. for 2hours. The reaction mixture was twice taken up and concentrated from 5ml of toluene, once from 5 ml of MeOH and once from 5 ml of hexanes toafford 51 mg (45%) of compound 138 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 427.21;C₂₄H₄₀N₂NaO₃, 405.25; C₂₄H₄₁N₂O₃.

Example 17 3-Amino-6,7-hydroxy-17-dimethyl Unsaturated Steroid

[0391] Any compounds related to compounds 88 or 89 can undergorearrangement using the method shown in Scheme 16. Treatment of compound88 with a 50° C. solution of hydrochloric acid in methanol and waterremoved the acetonide protecting group, facilitated migration of the18-methyl group to C17, and formed the ammonium chloride salt 139.Treatment of compound 89 with the same conditions also gave compound139. Example compounds 140-148 were synthesized using the method shownin Scheme 16 (see Table 4).

[0392] Synthesis of Compound 139

[0393] A solution of compound 88 (300 mg, 0.834 mmol), 4 drops ofconcentrated HCl, 2 ml of methanol and 2 ml of water was heated at 50°C. for 72 hours. The reaction mixture was concentrated and the residuewas twice taken up in 5 ml of methanol and concentrated. The residue wastaken up in 2 ml of methanol, diluted with 15 ml of acetone andconcentrated. The residue was triturated in 5 ml of acetone, filteredand dried to give 286 mg (96%) of compound 139 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)320.20; C₂₀H₃₄NO₂.

[0394] Synthesis of Compound 139

[0395] Using the same procedure as described for the synthesis ofcompound 139 from compound 88, compound 89 was reacted to give 145 mg(77%) of compound 139 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.20; C₂₀H₃₄NO₂.

[0396] Synthesis of Compound 140

[0397] Using the procedure described for the synthesis of compound 99,the amine 88 (200 mg, 0.55 mmol) was reacted with m-tolualdehyde (90 μl,0.61 mmol). Purification using radial chromatography eluting with 5%MeOH/EtOAc gave 127 mg of amine intermediate. A solution of theintermediate amine, 4 drops of concentrated HCl, 1 ml of MeOH and 1 mlof water was heated at 50° C. for 20 hours. The reaction mixture wastaken up and concentrated thrice from 5 ml of MeOH and once from 5 ml ofacetone to give 74 mg (30%/o) of compound 140 as an off-white foam.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 424.24; C₂₈H₄₂NO₂.

[0398] Synthesis of Compound 141

[0399] Using the procedure described for the synthesis of compound 99,the amine 88 (200 mg, 0.55 mmol) was reacted with3,4-difluorobenzaldehyde (67 μl, 0.61 mmol). Purification using radialchromatography eluting with 30% EtOAc/hexanes gave 88 mg of amineintermediate. A solution of the intermediate amine, 4 drops ofconcentrated HCl, 1 ml of MeOH and 1 ml of water was heated at 50° C.for 20 hours. The reaction mixture was taken up and concentrated thricefrom 5 ml of MeOH and once from 5 ml of acetone to give 73 mg (28%) ofcompound 141 as an off-white foam. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 446.42; C₂₇H₃₈F₂NO₂.

[0400] Synthesis of Compound 142

[0401] Using the procedure described for the synthesis of compound 59,the amine 49 (200 mg, 0.55 mmol) was reacted with3,4-dimethoxybenzaldehyde (70 μl, 0.61 mmol). Purification using radialchromatography eluting with 40% EtOAc/hexanes gave 67 mg of amineintermediate. A solution of the intermediate amine, 4 drops ofconcentrated HCl, 1 ml of MeOH and 1 ml of water was heated at 50° C.for 20 hours. The reaction mixture was taken up and concentrated thricefrom 5 ml of MeOH and once from 5 ml of acetone to give 43 mg (16%) ofcompound 142 as yellow solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 470.27; C₂₉H₄₄NO₄.

[0402] Synthesis of Compound 143

[0403] A solution of compound 28 (200 mg, 0.540 mmol), 4 drops ofconcentrated HCl, and 3 ml of water was heated at 50° C. for 72 hours.The reaction mixture was concentrated and the residue was twice taken upin 5 ml of methanol and concentrated. The residue was taken up in 3 mlof methanol, diluted with 20 ml of acetone and concentrated. The residuewas triturated in 10 ml of acetone, filtered and dried to give 179 mg(90%) of compound 143 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 334.20; C₂₁H₃₆NO₂.

[0404] Synthesis of Compound 144

[0405] Titanium(IV) isopropoxide (270 μl, 0.92 mmol) was added to a roomtemperature solution of the ketone 108 (250 mg, 0.67 mmol), aniline (56μl, 0.61 mmol) and 1.5 ml of THF under argon. After 12 hours a solutionof NaBH₄ (65 mg, 1.7 mmol) in 2.3 ml of EtOH was added and the reactionwas continued for another 8 hours. The reaction was quenched by theaddition of 0.5 ml of water and filtered to remove a white precipitate.The solution was loaded onto a column of 600 mg of MP-TsOH resin andeluted with 9 ml of MeOH then 9 ml of 2 M NH₃ in MeOH. The NH₃/MeOHfraction was concentrated and the residue was taken up in 4 ml THF andtreated with 500 mg of PS-benzaldehyde resin and filtered to remove anyresidual aniline. The solution was concentrated and the residue wastaken up in 2 ml of 9:1 THF and water and 100 μl of concentrated HCl.After stirring at room temperature overnight the reaction mixture wasconcentrated. The residue was triturated in 1 ml of cyclohexane,filtered and dried to afford 62 mg of compound 144 as a white solid.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 410.03; C₂₇H₄₀NO₂.

[0406] Synthesis of Compound 145

[0407] Using the procedures described for the synthesis of compound 144,compound 108 (250 mg, 0.67 mmol) was reacted with3-(trifluoromethyl)aniline. The intermediate product was purified usingradial chromatography and then reacted with 100 μl of concentrated HClin 2 ml of 9:1 THF and water. After stirring at room temperatureovernight the reaction mixture was concentrated and the residue wastriturated in 1 ml of cyclohexane, filtered and dried to afford 23 mg ofcompound 145 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 477.94; C₂₈H₃₉F₃NO₂.

[0408] Synthesis of Compound 146

[0409] Titanium(W) isopropoxide (216 μl, 0.73 mmol) was added to a roomtemperature solution of the ketone 108 (200 mg, 0.54 mmol), benzylamine(53 μl, 0.49 mmol) and 1.2 ml of THF under argon. After 12 hours asolution of NaBH₄ (52 mg, 1.4 mmol) in 1.7 ml of EtOH was added and thereaction was continued for another 6 hours. The reaction was quenched bythe addition of 1 ml of water and filtered to remove a whiteprecipitate. The solution was diluted with 70 ml of CH₂Cl₂, washed with10 ml of water and 20 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification using radial chromatography elutingconsecutively with 20% EtOAc/hexanes, EtOAc and 95:5:2 CH₂Cl₂/MeOH/Et₃Nafforded 127 mg of 3α-amine intermediate and 26 mg of 3β-amineintermediate. A solution of the 127 mg of 3α-amine intermediate, 1 ml of9:1 THF and water and 0.1 ml of concentrated HCl was stirred at roomtemperature overnight. The reaction mixture was concentrated, theresidue was taken up in 5 ml of MeOH and concentrated. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to afford 118 mg(95%) of compound 146 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 424.20; C₂₈H₄₂NO₂.

[0410] Synthesis of Compound 147

[0411] A solution of the 26 mg of 3β-amine intermediate, the synthesisof which was described under the synthesis of compound 146, 1 ml of 9:1THF and water and 0.1 ml of concentrated HCl was stirred at roomtemperature overnight. The reaction mixture was concentrated, theresidue taken up in 5 ml of MeOH and concentrated. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to afford 26 mg(100%) of compound 147 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 424.21; C₂₈H₄₂NO₂.

[0412] Synthesis of Compound 148

[0413] A solution of compound 78 (63 mg, 0.18 mmol), 4 drops ofconcentrated HCl, 1 ml of methanol and 1 ml of water was heated at 50°C. for 48 hours. The reaction mixture was concentrated and the residuewas twice taken up in 5 ml of methanol and concentrated. The residue wastaken up in 2 ml of hexanes, concentrated and dried for 2 hours using anAbderhalden drying apparatus with refluxing acetone to give 69 mg (100%)of compound 148 as a white solid. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂. TABLE 1

Calcium (% Com- 3α- or Hex IC₅₀ inhibition at pound 3β-NH₂ R₁ R₂ R₃ HA(μM) 20 μM) 49 α CH₃ H H HCl 11.0 26.8 53 α H H H AcOH 17.6 −0.8 54 α HH H HCl 16.1 19.8 64 α CH₃ F H HCl 14.4 6.7 69 α H CO₂CH₃ H AcOH 21.211.2 28 β CH₃ H H HCl 6.8 29.0 83 β CH₃ HG H AcOH 7.7 30.3 89 β H H HAcOH 11.5 15.0 95 β CH₃ F H HCl 13.5 11.2 78 α H H CH₃ none 20.1 5.3 79α H H CH₃ HCl 18.4 9.1 80 α H H CH₃ AcOH 10.7 7.4

[0414] TABLE 2

Calcium (% Com- 3α- or Hex IC₅₀ inhibition at pound 3β-NH₂ R₁ R₂ HA (μM)20 μM) 70 α O O AcOH ND 7.0 72 α OH H AcOH ND 10.3 96 β CH₃CH₂ H HCl 7.847.9 97 α CH₃CH₂ H HCl 9.3 43.0

[0415] TABLE 3

Calcium (% Com- 3α- or Hex IC₅₀ inhibition at pound 3β-NH₂ R₁ R₂ R₃ (μM)20 μM) 131 α H H CH₃CO 14.1 9.9 132 α H H C₆H₅CO 10.0 19.6 133 α H H(CH₃)₂CHSO₂ 15.9 13.8 134 α H H C₆H₅SO₂ 16.7 25.1 135 β CH₃ H

15.1 37.3 137 α H H C₆H₅NHCO 14.8 13.0 138 α H H CH₃(CH₂)₂NHCO 15.0 9.0

[0416] TABLE 4

Calcium Hex (% Com- 3α- or IC₅₀ inhibition pound 3β-NH₂ R₁ R₂ R₃ R₄ (μM)at 20 μM) 139 β CH₃ CH₃ H H 10.3 26.3 140 α CH₃ CH₃ H 3-(CH₃)C₆H₄CH₂10.2 21.5 141 α CH₃ CH₃ H 3,4-(F)₂C₆H₃CH₂ 9.4 36.3 142 α CH₃ CH₃ H3,4-(CH₃O)₂C₆H₃CH₂ 15.9 13.8 143 β CH₃ CH₃CH₂ H H 8.6 22.6 144 α and βCH₃ CH₃CH₂ H C₆H₅ 6.9 13.0 145 α and β CH₃ CH₃CH₂ H 3-(CF₃)C₆H₄ 22.0−0.2 146 α CH₃ CH₃CH₂ H C₆H₅CH₂ 9.0 41.7 147 β CH₃ CH₃CH₂ H C₆H₅CH₂ 18.136.3 148 α CH₃ CH₃ CH₃ H 29.2 6.6

[0417] TABLE 5

Hex Calcium (% Com- IC₅₀ inhibition at pound R HA (μM) 20 μM) 110

AcOH 10.9 39.7 111

HCl 9.3 53.1 113 CH₃NH HCl 9.8 45.0 114 CH₃(CH₂)₂NH HCl 10.2 37.9 115CH₃(CH₂)₄NH HCl 11.1 40.0 116 C₅H₉NH AcOH 9.6 60.9 117 C₆H₁₁NH AcOH 6.642.2 118

AcOH 11.0 41.8 119 CH₃(CH₂)₂NH(CH₂)₂NH 2HCl 6.1 54.5 120 (CH₃)₂N(CH₂)₂NH2HCl 9.1 44.0 121

AcOH 14.4 42.4 122 HOCH₂CH₂NH HCl 12.3 31.4 123 HOCH₂(CH₂)₄NH HCl 16.712.8 124 HOCH₂CH₂NHCH₂CH₂NH 2HCl ND 21.0 125 3-(CH₃)C₆H₄NH₂ HCl 39.4 5.7126 4-(HO)C₆H₄NH HCl 9.7 45.0 127 4-(H₂NSO₂)C₆H₄NH HCl 10.1 29.4 128

AcOH 10.5 41.0 129

2AcOH N/A 13.8

[0418] TABLE 6 Calcium (% Hex IC₅₀ inhibition at Compound R HA (μM) 20μM) 100 4-((CH₃)₂CH)C₆H₄ AcOH 18.2 33.2 101 2-(F)C₆H₄ AcOH 10.3 16.3 1023-(CF₃)C₆H₄ AcOH 10.0 9.5 103 2-(CH₃O)C₆H₄ AcOH 11.1 23.7 1044-(CF₃O)C₆H₄ AcOH 9.5 30.3 105 3-(C₆H₅O)C₆H₄ AcOH 22.5 10.2 1063-(NO₂)C₆H₄ none 8.8 8.1 107 3-C₅H₄N 2HCl 17.2 11.6

UTILITY EXAMPLES Example A Effect of Selected Compounds onAllergen-Induced Lung Inflammation

[0419] The ability of a compound to inhibit the allergen-inducedaccumulation of inflammatory cells such as eosinophils and neutrophilsin the lavage fluid obtained from sensitized animals is indicative ofthat compound's anti-asthma activity. In particular, this model systemis useful in the evaluation of the effects of a test compound in thetreatment of the late phase response of asthma, when lung inflammationand the second phase of bronchoconstriction is apparent. The test isconducted as follows.

[0420] Male Brown Norway rats are sensitized to ovalbumin by singleintraperitoneal injection of 1 mg ovalbumin adsorbed to 100 mg AM(OH)₃(alum) in 1 ml sterile saline (saline control rats receive only sterilesaline) on day 1, and allowed to sensitize until day 21. Test compoundsare given orally q.d. for three days prior to challenge (days 19, 20,21), and one day post challenge (day 22), with the third dose given 2hours before challenge, and the fourth day dose given 24 hours afterchallenge (volume=300 μI/dose). Rats are challenged with 0.5% ovalbuminin saline generated using a Devillbis nebulizer for 60 min on day 21.

[0421] Forty-eight hrs after challenge, animals are sacrificed with anoverdose of intraperitoneally-delivered sodium pentobarbitol and thelungs are lavaged with cold 2×7 ml phosphate buffered saline. Therecovered lavage fluid is placed on ice. The bronchoalveolar lavagefluid is centrifuged and the supernatant removed. The pellet isresuspended in phosphate buffered saline at 4° C. Cytospins are preparedand stained for differentiation and enumeration of cell types.

[0422] The protective effects of the various test compounds on allergeninduced lung inflammation are summarized in Tables 7 and 8. The doseresponse activity of select compounds is shown in Table 9. Test compoundwas administered in 300 μl corn oil (Tables 7 and 8) or water (Table 9),which were used as vehicles. Control animals received 300 μl corn oil orwater alone, i.e., no drug. Values in Tables 7, 8, and 9 representpercent inhibition of leukocyte accumulation relative to controlanimals. A negative value in Table 7, 8, or 9 indicates an exacerbationof the effect over the control animal. TABLE 7 EFFECT OF TEST COMPOUNDS(5 MG/KG/DAY FOR 4 DAYS, P.O.) ON OVALBUMIN-INDUCED ACCUMULATION OFINFLAMMATORY CELLS IN THE LUNG LAVAGE FLUID OBTAINED FROM SENSITIZEDBROWN NORWAY RATS % inhibition of % inhibition of % inhibition ofCompound cosinophils neutrophils lymphocytes 83 67 34 38 97 40 52 56 96−9 40 19 64 62 70 81 89 57 60 64 28 85 87 124 53 24 57 13 95 14 45 30 4952 36 73 135 20 58 107

[0423] TABLE 8 EFFECT OF SELECTED COMPOUNDS (1 MG/KG/DAY FOR 4 DAYS,P.O.) ON OVALBUMIN INDUCED ACCUMULATION OF INFLAMMATORY CELLS IN THELUNG LAVAGE FLUID) OBTAINED FROM SENSITIZED BROWN NORWAY RATS %inhibition of % inhibition of % inhibition of Analogue eosinophilsneutrophils lymphocytes 142 −131 −1 −42 54 −82 −19 −7 107 −122 23 −28124 −55 −76 −26 129 −296 −114 −71 146 8 24 −27 147 40 58 36 131 16 57 21138 −52 35 33 133 −43 40 28

[0424] TABLE 9 DOSE DEPENDENT EFFECT OF SELECTED COMPOUNDS (MULTIPLEDOSING, 4 DAYS QD, P.O.) ON OVALBUMIN-INDUCED ACCUMULATION OF INFLAMMA-TORY CELLS N THE LUNG LAVAGE FLUID OBTAINED FROM SENSITIZED BROWN NORWAYRATS % inhibition of % inhibition of % inhibition of Analogueeosinophils neutrophils lymphocytes mg/kg 1 0.3 0.1 0.03 1 0.3 0.1 0.031 0.3 0.1 0.03 89 71 80 35 — 66 72 25 — 6 70 2 — 28 60 71 29 5 41 66 −10−26 70 85 50 −5.00 139 42 41 46 — 63 62 42 — 56 34 19 — 143 48 29 28 —64 25 57 — −22 −37 −43 —

Example B Effect of Compound 83 on Irritant-Induced Mouse Ear Edema

[0425] A number of mice are uniquely identified by placing a mark withan indelible marker on their tail. Mice are dosed orally with 15 mg/kgtest compound in 100 μl of 45% β-cyclodextrin in saline. Mice arebriefly anaesthesized with 2% halothane, and 2 μg of phorbol12-myristate 13-acetate in 25 μl of acetone is applied to the inner andouter sides of the left ear of the mouse. Acetone is applied to theright ear of the mouse in the same manner to serve as a vehicle control.Control animals receive the same treatment but without any testcompound. After 3 hours, mice are sacrificed by cervical dislocation,and a standard sized biopsy is excised from the ears and weighed to thenearest {fraction (1/10)}th of a mg. Data are analyzed by taking thedifference of each left ear from the right ear, and then calculating the% inhibition of edema by (((mean Rx/mean irritant))×100)-100.

[0426] The compounds of the present invention demonstrate protectiveeffects on irritant induced mouse ear edema. For example, compound 83inhibits irritant-induced mouse ear edema by 38% compared to controlanimals.

Example C Effect of Compounds on the Release of Hexosaminidase from aRat Mast Cell Line (RBL-2H3)

[0427] The anti-allergic effects of a compound of the present inventionwas evaluated by measuring its effect on antigen-induced secretion ofhexosaminidase from a passively sensitized rat mast cell line. Theability of a test compound to inhibit the release of mast cell granulecontents, e.g., histamine and hexosaminidase, is indicative of theanti-allergy and/or anti-asthma activity of the compound. Hexosaminidaseis released from the mast cell granule along with histamine and othermediators during antigen challenge. The test is performed as follows.

[0428] RBL-2H3 cells are grown in culture and passively sensitized for 1hour at 37° C. to dinitrophenol (DNP) using anti-human-DNP (IgE). Cellsare incubated with test compound for 30 minutes at 37° C. and stimulatedwith 0.5 μg/ml DNP-HSA (antigen) for 30 minutes. Aliquots of thesupernatant are removed and used to measure the amount of hexosaminidasereleased during challenge with the antigen. The amount of hexosaminidasepresent in the supernatant is determined colorimetrically by monitoringthe enzymatic metabolism of p-nitrophenyl-N-acetyl-β-D-glucosaminide(p-NAG) over a period of 60 minutes at 405 nm. The effect of each testcompound is determined as a percentage of the antigen-induced response(minus background release) obtained in the presence of DMSO alone. Thesevalues are used to determine the degrees of inhibition ofantigen-induced hexosaminidase release from the cells.

[0429] The compounds of the present invention demonstrate the ability toinhibit hexosaminidase release in response to antigen stimulation.Compounds were tested at 0.3, 3, 10 and 30 μM, and the IC₅₀ calculated.This data is summarized in Tables 1-6. For example, compound 119 at 6.1μM inhibits hexosaminidase release by 50% in response to antigenstimulus.

Example D Metabolic Stability of Selected Compounds in Human S9Fractions

[0430] The therapeutic effectiveness of a test compound can be oftendirectly related to its metabolic stability in vivo. The majority ofknown, marketed drugs are metabolized by a group of enzymes known asP450 enzymes. The S9 fraction of human liver contains all the P450enzymes and also cytoplasmic enzymes that may be involved in themetabolism of new chemical entities. Metabolism in vitro using human S9fractions is a standard assay to evaluate relative metabolic stabilityof new compounds. The test is performed as follows.

[0431] Reagents are thawed on ice and combined to make up a Master Mixas follows: Potassium phosphate (100 mM, pH 7), G6P (0.25 mM), G6PDH (2U/ml), NADPH (1 mM), UDP (0.25 mM), APPS (0.25 mM), and S9 fraction (2mg/ml). A volume of 498 μl of the Master mix is dispensed into eachmicrocentrifuge tube. A volume of 2 μl of 2.5 mM test compound (finalconcentration of 10 μM) is dispensed into the center of the lid of theappropriate tube, and the lids gently closed to prevent mixing.Synchronous combination of test compound and master mix is achieved bysimultaneous ten times inversion of the tubes, which are then incubatedat 37° C. and shaken at 150 rpm for the appropriate incubation times.While incubation is in progress, all 0 time samples are mixedindividually by three times inversion followed by the immediate additionof 500 μl ice cold acetonitrile with three times inversion to stop thereaction. Immediately after each 15 minutes and 30 minutes incubation iscomplete, ice cold acetonitrile is added to each tube and the tubesmixed by three times inversion. All sample tubes are incubated at −80°C. for a minimum of 15 minutes, thawed and remixed by inversion.Aliquots of 650 μl are transferred to Chromatographic Specialtiesmicro-system spin filter system tubes (0.2 μm nylon membrane, C618505)and centrifuged at 13,000 rpm for 48 seconds. The sample filtrates arestored at −80° C.

[0432] Sample filtrates are analyzed on LCMS, and percent remainingafter 15 and 30 minute incubation is calculated relative to the 0 minuteincubation.

[0433] The metabolic stability of various compounds are summarized inTable 10 as the percent remaining after 15 or 30 minutes incubation withhuman liver S9 fractions. TABLE 10 METABOLIC STABILITY OF SELECTEDCOMPOUNDS AFTER 15 AND 30 MINUTES INCUBATION WITH HUMAN LIVER S9FRACTIONS SHOWN AS THE PERCENT REMAINING OF STARTING CONCENTRATION AFTER0 MINUTES INCUBATION Compound % remaining after 15 min % remaining after30 min 83 92 ± 19 91 ± 21 97 94 ± 10 89 ± 12 96 92 ± 19 94 ± 21 89 100 ±17  98 ± 16 28 100 ± 6  104 ± 12  49 93 ± 6  85 ± 8  64 104 ± 4  88 ± 5 139 109 ± 26  100 ± 10  143 100 ± 3  99 ± 10 146 82 ± 6  63 ± 9  107 84± 5  62 ± 6  142 80 ± 11 55 ± 2  69 73 ± 24 63 ± 6  104 53 ± 10 56 ± 18141 100 ± 9  26 ± 4  134 85 ± 54 36 ± 15 137 93 ± 31 71 ± 17 138 71 ± 4 65 ± 65 132 60 ± 17 40 ± 9  79 67 ± 6  61 ± 14

Example E Solubility of Selected Compounds in Physiologically CompatibleFormulations

[0434] Compounds of the present invention exhibit good water solubility.For example, compound 83 is soluble in water at 225 mg/ml. Compound 83substituted with a hydroxyl at C3 has a solubility in water of less than60 μg/ml. Compounds 28 and 89 have solubilities at room temperature of˜30 mg/ml, which can be significantly increased by heating. Thisunexpected finding indicates that these 3-amino compounds should bereadily formulated into therapeutic compositions.

Example F Effect of Selected Test Compounds on Antigen-Induced CalciumFlux

[0435] Elevation of cytoplasmic calcium concentration is a common andcrucial event which follows the activation of many types of cell surfacereceptors. Increases in intracellular calcium that occur followingagonist activation of inositol lipid hydrolysis are the results ofcalcium release from the endoplasmic reticulum and the influx of calciumthrough the plasma membrane. Increases in cytosolic calciumconcentration are involved in many important cellular responses in theinflammatory process including adhesion, motility, gene expression,proliferation, and degranulation. Changes in intracellular calciumconcentrations can affect both short and long term cellular responses.An assay method to evaluate a test compound's impact on calcium flux isprovided as follows.

[0436] Jurkat clone E6.1 cells grown in RPMI medium supplemented with10% FBS and 2 mM L-Glutamine are transferred to 50 mL conical tubes andcentrifuged for 5 minutes at 900 RPM to form a cell pellet. Theresulting supernatants are discarded and each pellet is washed in 10 mLHBSS. Cell suspensions are accumulated and centrifuged for 5 minutes at900 RPM to form a cell pellet. The resulting supernatant is discardedand the pellet is resuspended in HBSS at 1×10⁷cells/mL. The cellsuspension is transferred to a 20 mm petri dish and incubated at 37° C.,5% CO₂ for 20 minutes.

[0437] One volume of Fura 2AM is mixed to one volume of detergentPluronic F127. Cells are labeled with 4 μL of probe solution to each mLof cell suspension. The petri dish is wrapped in aluminum foil toprotect from light and placed on a plate shaker for 30 minutes at roomtemperature.

[0438] The following steps are done in the laminar flow hood with thefluorescent lights turned off. The petri dish is removed from the shakerand the labeled cell suspension is transferred to a 15 mL conical tubeand washed twice with HBSS as above. The cell pellet is resuspended in12 mL HBSS and left wrapped in foil for 30 minutes at room temperature.The labeled cell suspension is aliquoted (100 μL/well) into a Dynex 96well white opaque tissue culture plate. 50 μL of each test sample isadded to appropriate wells and are incubated together for 10 minutes at37° C. in the Wallac 1920 Victor™ plate reader (test samples areprepared in HBSS at 20 mM, final concentration is 20 μM). Selectedcompounds were tested for dose related activity. Test samples andactivator (anti-CD3 mAb at final concentration of 4 μg/mL, PharMingen)are added manually (50 μL), such that the minimum time to acquire thefirst data point after stimulation is approximately 30 seconds. Thecalcium influx response to anti-CD3 mAb is measured as an “end point”assay. The entire plate is read in 100 seconds using a kinetic of 1second per well.

[0439] The plate is read before the addition of anti-CD3 to monitornon-specific effect of samples/drugs. Fluorescence emission is measuredat 510 nm with excitation alternating between 340 and 380 every secondusing an excitation/emission filter pair. Data from these dualwavelengths are represented as a ratio of 2 excitations wavelengths.This ratio is independent of intracellular dye and cell concentrations,enabling real comparison between experiments.

[0440] The effect of selected compounds on calcium flux in antigenchallenged Jurkat clone E6.1 cells is summarized in Tables 1-6. Forexample, compound 116 inhibited calcium by 60.9% at 20 μM. The IC₅₀ forcompound 119 is less than or equal to 10 μM when dose response activitywas examined. This demonstrated substantial effect on calcium flux wouldbe beneficial in any disease pathology for which calcium is asignificant second messenger or effector molecule, including but notlimited to ischemia/reperfusion injury such as stroke or myocardialinfarction, inflammatory diseases such as asthma or allergy, neural ormuscular disordcrs such as Parkinson's disease or epilepsy, cardiacarrhythmias, or hypertension.

Example G Effect of Selected Compounds on Allergen-Induced Changes inLung Function

[0441] In asthma, the early response of the airways to allergenchallenge is characterized by an immediate bronchoconstriction whichpeaks 20-30 min after exposure to the stimuli, and which normallyresolves after approximately 2 hours. Anti-inflammatories are notgenerally active bronchodilators and are not very effective in thecontrol of acute asthmatic bronchoconstriction. This results in the needfor combination therapy to treat both the bronchoconstriction and theinflammation.

[0442] Cam-Hartley guinea pigs were sensitized to ovalbumin (OA) ingroups of 5-6 by exposure to an aerosolized solution of 1% OA in salinefor 15 min on 2 consecutive days via a DeVilbiss nebulizer, with anadditional single intradermal injection of 3 μg OA in saline on Day 1.Animals were found to be at peak sensitivity to the antigenapproximately 14 days after the initial exposure. On Day 14, the animalswere initially anaesthetized with ketamine (50 mg/kg i.p.) and xylazine(10 mg/kg i.p.), weighed, and then maintained on 1% halothane deliveredvia a nose cone. The left carotid artery was cannulated with PE90 tubingcontaining 200 U/ml heparin in saline. A tracheostomy was performed anda fluid-filled cannula (PE 160) was inserted approximately 7 cm into theesophagus. The animal was positioned in a plethysmograph and the tracheaattached to a fixed stainless steel tracheal tube in the body box. Thecarotid cannula was attached to a pressure transducer for monitoring ofblood pressure and heart rate. The guinea pig was paralyzed withpancuronium bromide (0.8 mg/kg) and ventilated with air at a frequencyof 60 Hz and tidal volume of 3 ml using a Harvard small animalventilator.

[0443] Data was collected for 20 sec periods at a sampling rate of 100Hz on a computer-linked physiological recording system using DIRECphysiological software and analyzed using ANADAT software. Pulmonaryresistance and dynamic lung compliance values were obtained from thevolume, flow and pressure signals according to the method of VonNeeguard & Wirz (1927), using an isovolumetric multi-point regressionmodel for analysis (Ludwig, Robatto, et al. 1991), and calculated asabsolute changes in lung resistance (RL; cm H₂O/ml/s) or lung compliance(CDYN; ml/cmH₂O). Volume and pressure signals were calibrated beforeeach set of experiments following standard procedures.

[0444] Several lung function measurements were obtained over a 5-10minute period to ensure a steady baseline, and then the animal waschallenged with OA (2% in saline) administered in 6 tidal breaths as anebulized aerosol at a flow rate of 5 L/min. Pulmonary andcardiovascular function was continually monitored throughout theexperiment, although data was collected at specific time-points afterantigen challenge (10 s, 1, 2, 3, 4, 5, 10, 20, and 30 min).

[0445] Test compounds were administered under light halothane anesthesiaby oral gavage (0.1-1.0 mg/kg/day q.d.) in 300 μl polyethyleneglycol-200 for 4 days prior to challenge with the final doseadministered 2 hours prior to antigen challenge.

[0446] The protective effects of select test compounds on allergeninduced bronchoconstriction are summarized in FIGS. 4 and 5. Theduration of activity of Compound 89 is shown in FIGS. 6 and 7. Data ispresented as mean±standard error of the mean. The inhibition of thebronchoconstriction by the test compounds would be beneficial in anydisease where acute smooth muscle constriction in response to allergenchallenge is manifest, such as asthma and allergy.

[0447] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually incorporated by reference. For example, the book inComprehensive Organic Transformations, A Guide to Functional GroupPreparations, Second Edition, Richard C. Larock, John Wiley and Sons,Inc., 1999, and particularly the references cited therein, isincorporated herein by reference for all purposes.

[0448] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound of the formula

and pharmaceutically acceptable salts, solvates, stereoisomers andprodrugs thereof, in isolation or in mixture, where independently ateach occurrence: R¹ and R² are selected from hydrogen, oxygen so as toform nitro or oxime, amino, —SO₃—R, and organic groups having 1-30carbons and optionally containing 1-6 hetero atoms selected fromnitrogen, oxygen, phosphorous, silicon, and sulfur, where R² may be adirect bond to numeral 3, or R¹ and R² may, together with the N to whichthey are both bonded, form a heterocyclic structure that may be part ofan organic group having 1-30 carbons and optionally containing 1-6heteroatoms selected from nitrogen, oxygen and silicon; or R¹ may be a 2or 3 atom chain to numeral 2 so that —N—R¹— forms part of a fusedbicyclic structure to ring A; R³ and R⁴ are selected from direct bondsto 6 and 7 respectively so as to form carbonyl groups, hydrogen, or aprotecting group such that R³ and/or R⁴ is part of hydroxyl or carbonylprotecting group; numerals 1 through 17 each represent a carbon, wherecarbons at numerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independentlysubstituted with (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵)_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or (b)) two of the following, which are independentlyselected: —X, —N(R¹)(R²), —R⁵ and —OR⁶; and where carbons at numerals 5,8, 9, 10, 13 and 14 may be independently substituted with one of —X,—R⁵, —N(R¹)(R²) or —OR⁶; in addition to the —OR³ and —OR⁴ groups asshown, each of carbons 6 and 7 may be independently substituted with oneof —X, —N(R¹)(R²), —R⁵ or —OR⁶; each of rings A, B, C and D isindependently fully saturated, partially saturated or fully unsaturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded; R⁶ is H or aprotecting group such that —OR⁶ is a protected hydroxyl group, wherevicinal —OR⁶ groups may together form a cyclic structure that protectsvicinal hydroxyl groups, and where geminal —OR⁶ groups may together forma cyclic structure that protects a carbonyl group; and X representsfluoride, chloride, bromide and iodide.
 2. A compound of claim 1 whereinnumerals 1 through 16 each represent a carbon, where carbons at numerals1, 2, 4, 11, 12, 15 and 16 may be independently substituted with (a) oneof: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵), —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and—(O(C(R⁵)(R⁵))_(n)O)— wherein n ranges from 1 to about 6; or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), —R⁵ and—OR⁶; and numeral 17 represents a carbon substituted with (a) one of:═C(R^(5a))(R^(5a)), ═C═C(R^(5a))(R^(5a)), and—C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)— wherein n ranges from 1 toabout 6; or (b) two of the following, which are independently selected:—X, —N(R¹)(R²), and —R^(5a); where R^(5a) at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, silicon and sulfur; where twogeminal R⁵ groups may together form a ring with the carbon atom to whichthey are both bonded.
 3. A compound of claim 2 wherein R^(5a) at eachoccurrence is independently selected from C₁₋₃₀ hydrocarbon, C₁₋₃₀halocarbon, Cl₃₀ hydrohalocarbon, H, and X.
 4. A compound of claim 2wherein R^(5a) at each occurrence is independently selected from C₁₋₁₀hydrocarbon, C₁₋₁₀ halocarbon, C₁₋₁₀ hydrohalocarbon, H, and X.
 5. Acompound of any of claims 1-4 wherein R¹ and R² are selected fromhydrogen, oxygen so as to form nitro or oxime, amino, —SO₃—R, andorganic groups having 1-30 carbons and optionally containing 1-6heteroatoms selected from oxygen, phosphorous, silicon, and sulfur,where R² may be a direct bond to numeral 3, or R¹ and R² may, togetherwith the N to which they are both bonded, form a heterocyclic structurethat may be part of an organic group having 1-30 carbons and optionallycontaining 1-6 heteroatoms selected from oxygen and silicon; or R¹ maybe a 2 or 3 atom chain to numeral 2 so that —N—R¹— forms part of a fusedbicyclic structure to ring A.
 6. A compound of any of claims 1-5 whereincarbons at numerals 1, 2, 4, 11, 12, 15 and 16 are each substituted withtwo hydrogens unless said carbon is part of an unsaturated bond; carbonsat numerals 5, 8, 9 and 14 are each substituted with one hydrogen unlesssaid carbon is part of an unsaturated bond; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond.
 7. A compound of any ofclaims 1-6 wherein carbons at numerals 1, 2, 4, 11, 12, 15 and 16 areeach substituted with two hydrogens; carbons at numerals 5, 8, 9 and 14are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond.
 8. A compound of claim1 wherein R¹ and R² are hydrogen; R³ and R⁴ are selected from directbonds to 6 and 7 respectively so as to form carbonyl groups, hydrogen,or a protecting group such that R³ and/or R⁴ is part of hydroxyl orcarbonyl protecting group; and in addition to the —OR³ and —OR⁴ groupsas shown, each of carbons 6 and 7 is substituted with hydrogen unlessprecluded because —OR³ or —OR⁴ represent a carbonyl group; carbons atnumerals 1, 2, 4, 11, 12, 15 and 16 are each substituted with twohydrogens unless said carbon is part of an unsaturated bond; carbons atnumerals 5, 8, 9 and 14 are each substituted with one hydrogen unlesssaid carbon is part of an unsaturated bond; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)R⁵(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n ranges from1 to about 6; or (b) two of the following, which are independentlyselected: —X, —N(R¹)(R²), —R⁵ and —OR⁶; each of rings A, B, C and D isindependently fully saturated, partially saturated or fully unsaturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded; R⁶ is H or aprotecting group such that —OR⁶ is a protected hydroxyl group, wherevicinal —OR⁶ groups may together form a cyclic structure that protectsvicinal hydroxyl groups, and where geminal —OR⁶ groups may together forma cyclic structure that protects a carbonyl group; and X representsfluoride, chloride, bromide and iodide.
 9. A compound of claim 8 whereinR¹ and R² are hydrogen; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of the following,which are independently selected: —X, —N(R¹)(R²), and —R⁵; each of ringsA, B; C and D is independently fully saturated or partially saturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀hydrocarbons, halocarbons and halohydrocarbons; and X representsfluoride, chloride, bromide and iodide.
 10. A compound of claim 9wherein R¹ and R² are hydrogen; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵); or (b) two of —R⁵; each of rings A, B, C and Dis independently fully saturated or partially saturated; and R⁵ at eachoccurrence is independently selected from H and C₁₋₁₀ hydrocarbons. 11.A compound of any one of claims 1-10 of the formula


12. A compound of any one of claims 1-10 of the formula


13. A compound of any one of claims 1-10 of the formula


14. A compound of any one of claims 1-10 of the formula


15. A compound of any one of claims 1-10 of the formula


16. A compound of any one of claims 1-10 of the formula


17. A compound of claim 1 wherein 17 is substituted with ═C(R⁵)(R⁵) andR⁵ is selected from hydrogen, halogen, C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, and—CO₂—C₁₋₆alkyl.
 18. A compound of claim 1 wherein 17 is substituted withC₁₋₆alkyl or C₁₋₆haloalkyl.
 19. A compound of claim 1 wherein 17 issubstituted with —OR⁶ or ═O, wherein R⁶ is hydrogen.
 20. A compound ofclaim 1 wherein R¹ is selected from —C(═O)—R⁷, —C(═O)NH—R⁷; —SO₂—R⁷;wherein R⁷ is selected from alkyl, heteroalkyl, aryl and heteroaryl. 21.A compound of claim 20 wherein R⁷ is selected from C₁₋₁₀hydrocarbyl. 22.A compound of claim 20 wherein R⁷ comprises biotin.
 23. A compound ofclaim 1 wherein (R¹)(R²)N— is selected from


24. A compound of claim 1 wherein R¹ is hydrogen and R² comprises acarbocycle.
 25. A compound of claim 24 wherein the carbocycle is phenyl.26. A compound of claim 25 wherein R² is selected from 3-methylphenyl;4-hydroxyphenyl; and 4-sulfonamidephenyl.
 27. A compound of claim 1wherein R¹ is hydrogen and R² comprises a C₁₋₁₀hydrocarbyl.
 28. Acompound of claim 1 wherein R¹ is hydrogen and R² is heteroalkyl.
 29. Acompound of claim 28 wherein R² is selected fromC₁₋₁₀alkyl-W—C₁₋₁₀alkylene-wherein W is selected from O and NH;HO—C₁₋₁₀alkylene-; and HO—C₁₋₁₀alkylene-W—C₁₋₁₀alkylene-where W isselected from O and NH.
 30. A compound of claim 1 wherein R¹ is hydrogenand R² is —CH₂—R⁷ wherein R⁷ is selected from alkyl, heteroalkyl, aryland heteroaryl.
 31. A compound of claim 30 wherein R⁷ is selected fromalkyl-substituted phenyl; halogen-substituted phenyl; alkoxy-substitutedphenyl; aryloxy-substituted phenyl; and nitro-substituted phenyl.
 32. Acompound of claim 1 wherein each of R¹ and R² is hydrogen.
 33. Acompound of claims 1 or 32 wherein each of R³ and R⁴ is hydrogen.
 34. Acompound of claims 32 or 33 where the carbon at numeral 17 issubstituted with (a) one of the following: C(R^(5a))(R^(5a)),═C═(R^(5a))(R^(5a)), and —C(R^(5a))(R^(5a))(C)(R^(5a))(R^(5a)))_(n)—wherein n ranges from 1 to about 6; or (b) two of the following, whichare independently selected: —X, —N(R¹)(R²), and —R^(5a); where R^(5a) ateach occurrence is independently selected from H, X, and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, silicon and sulfur;where two geminal R⁵ groups may together form a ring with the carbonatom to which they are both bonded.
 35. A compound of claim 1 wherein R³and R⁴ together form a ketal of the structure


36. A compound of claim 1 wherein —OR³ and —OR⁴ have the stereochemistryshown


37. A compound of claim 1 wherein —N(R¹)(R²) is in a salt form.
 38. Acompound of claim 1 wherein —N(R¹)(R²) is in a salt form and the salt isa halogen or acetate salt.
 39. A compound of claim 1 which is a prodragof the formula shown in claim
 1. 40. A compound of claim 1 andpharmaceutically acceptable salts, solvates, stereoisomers but notprodrugs thereof, in isolation or in mixture.
 41. A compound of claim 1wherein at least one of the carbons at numerals 10 and 13 aresubstituted with methyl.
 42. A compound of claim 1 wherein each of R¹and R² are independently selected from hydrogen and organic groupshaving 1-20 carbons and optionally containing 1-5 heteroatoms selectedfrom nitrogen, oxygen, silicon, and sulfur.
 43. A compound of claim 1wherein R¹ and R² are independently selected from hydrogen, R⁸, R⁹, R¹⁰,R¹¹ and R¹² where R⁸ is selected from alkyl, heteroalkyl, aryl andheteroaryl; R⁹ is selected from (8)_(r)-alkylene,(R⁸)_(r)-heteroalkylene, (R⁸)_(r)-arylene and (R⁸)_(r)-heteroarylene;R¹⁰ is selected from (R⁹)_(r)-alkylene, (R⁹)_(r)-heteroalkylene,(R⁹)_(r)-arylene, and (R⁹)_(r)-heteroarylene; R¹¹ is selected from(R¹⁰)_(r)-alkylene, (R¹⁰)_(r)-heteroaalene, (R¹⁰)_(r)-arylene, and(R¹⁰)_(r)-heteroarylene, R¹² is selected from (R¹¹)_(r)-alkylene,(R¹¹)_(r)-heteroalkylene, (R¹¹)_(r)-arylene, and(R¹¹)_(r)-heteroarylene, and r is selected from 0, 1, 2, 3, 4 and 5,with the proviso that R¹ and R² may join to a common atom so as to forma ring with the common atom.
 44. A compound of claims 1 or 43 wherein R³and R⁴ are selected from hydrogen and protecting groups such that R³and/or R⁴ is part of hydroxyl protecting group; carbons at numerals 1,2, 4, 11, 12, 15 and 16 are each substituted with two hydrogens unlesssaid carbon is part of an unsaturated bond; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen unless said carbon is partof an unsaturated bond; carbon at numeral 10 is substituted with methyl;carbon at number 13 is substituted with methyl unless it is part of anunsaturated bond; carbon at numeral 17 is substituted with (a) one of═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of —R⁵; each of rings A, B, Cand D is independently fully saturated or partially saturated; and R⁵ ateach occurrence is independently selected from H and C₁₋₁₀ hydrocarbons.45. A compound of claims 1, 43 or 44 wherein R¹ and R² are independentlyselected from hydrogen, R⁸, R⁹, R¹⁰, R¹¹ and R¹² where R⁸ is selectedfrom C₁₋₁₀alkyl, C₁₋₁₀heteroalkyl comprising 1, 2 or 3 heteroatoms,C₆₋₁₀aryl and C₃₋₁₅heteroaryl comprising 1, 2 or 3 heteroatoms; R⁹ isselected from (R⁸)_(r)—C₁₋₁₀alkylene, (R⁸)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁸)_(r)—C₆₋₁₀aylene and(R⁸)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹⁰ isselected from (R⁹)_(r)—C₁₋₁₀alkylene, (R⁹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁹)_(r)—C₆₋₁₀arylene, and(R⁹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹¹ isselected from (R¹⁰)_(r)—C₁₋₁₀alkylene, (R¹⁰)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹⁰)_(r)—C₆₋₁₀arylene, and(R¹⁰)_(r)—C₃₋₁₅heteroamylene comprising 1, 2 or 3 heteroatoms, R¹² isselected from (R¹¹)_(r)—C₁₋₁₀alkylene, (R¹¹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹¹)_(r)—C₆₋₁₀arylene, and(R¹¹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, and r isselected from 0, 1, 2, 3, 4 and 5, with the proviso that R¹ and R² mayjoin to a common atom so as to form a ring with the common atom.
 46. Acompound of claims 1, 43 or 44 wherein R¹ and R² are selected fromhydrogen, CH₃—, CH₃(CH₂)₂—, CH₃(CH₂)₄—, CH₃CO—, C₆H₅CO—(CH₃)₂CHSO₂—,C₆H₅SO₂—, C₆H₅NHCO—, CH₃(CH₂)₂NHCO—, CH₃(CH₂)₂NH(CH₂)₂—, (CH₃)₂N(CH₂)₂—,HOCH₂CH₂—, HOCH₂(CH₂)₄—, HOCH₂CH₂NHCH₂CH₂—, 3-(CH₃)C₆H₄—, 4-(HO)C₆H₄—,4-(H₂NSO₂)C₆H₄—, 4-((CH₃)₂CH)C₆H₄—CH₂—, 2-(F)C₆H₄—CH₂—,3-(CF₃)C₆H₄—CH₂—, 2—(CH₃O)C₆H₄—CH₂—, 4-(CF₃O)C₆H₄—CH₂—,3-(C₆H₅O)C₆H₄—CH₂—, 3-(NO₂)C₆H₄—CH₂—,

or R¹ and R² may join together with the nitrogen to which they are bothattached and form a heterocycle selected from:


47. A compound of claims 1 or 43 of the formula


48. A compound of claims 1 or 43 of the formula


49. A compound of claims 1 or 43 of the formula


50. A compound of claims 1 or 43 of the formula


51. A compound of claim 1 wherein R¹ is a 2, or 3 atom chain to numeral2 so that —N—R¹— forms part of a fused bicyclic structure to ring A, thecompound having the formula:

where Z represents 2 or 3 atoms, independently selected from C, N and Oso long as a stable structure results, and the ring including Z may besaturated or unsaturated.
 52. A compound of claim 51 selected from


53. A pharmaceutical composition comprising a compound of any of claims1-52 and a pharmaceutically acceptable carrier, excipient or diluent.54. A method of treating inflammation therapeutically comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 55. A method of treatinginflammation prophylactically comprising administering to a subject inneed thereof a prophylactically-effective amount of a compound of any ofclaims 1-52.
 56. A method of treating asthma comprising administering toa subject in need thereof a therapeutically-effective amount of acompound of any of claims 1-52.
 57. A method of treating allergicdisease including but not limited to dermal and ocular indicationscomprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.58. A method of treating chronic obstructive pulmonary diseasecomprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.59. A method of treating atopic dermatitis comprising administering to asubject in need thereof a therapeutically-effective amount of a compoundof any of claims 1-52.
 60. A method of treating solid tumours comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 61. A method of treatingAIDS comprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.62. A method of treating ischemia reperfusion injury comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 63. A method of treatingcardiac arrhythmias comprising administering to a subject in needthereof a therapeutically-effective amount of a compound of any ofclaims 1-52.